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User Manual Chirascan Series Spectrometers

1. 0 1303 0 3153 180 00 196 00 21200 22800 244 00 260 00 180 00 196 00 21200 22800 244 00 260 00 Wavelength nm Wavelength nm p Figure 5 11 An appropriate smooth left and an inappropriate one right Enter the number of degrees of smoothing required in the Window Size box This value will depend on the step resolution used during the measurement and the sharpness of the features in the spectrum The number should be as large as is possible without causing distortion of the spectrum Examination of the residual plot will show if any distortion has occurred If the noise is randomly distributed about zero the smoothing process has not caused any distortion and importantly you have the evidence to prove it An example of an appropriate smooth in which 5 degrees of smoothing were used and an inappropriate one in which 12 degrees of smoothing were used on the same data set are shown in Figure 5 11 The structure in the residuals that arises when 12 degrees of smoothing were used is apparent When you are satisfied with the smoothing process click OK Document 4207Q108C03 01 Page 93 of 133 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences eee eee eee eee eee eee eee eee 1 Click Unset Background 2 Select Smooth S 6 and Smooth r manipulation dialog box will appear as in Figure 5 12 Chirascan Series User Manual and click Remove Oth
2. 260 Chirascan Series User Manual Figure 5 50 Lysozyme spectrum after averaging background correction and smoothing CDNN requires a text file input format with wavelengths and associated CD values at 1 nm intervals It does not matter if you have measured the spectrum at a different step size the above spectra were measured at 0 5 nm steps because there is a conversion program that generates the correct input format Select File gt Save Selected Traces from the graphical display File menu Section 5 8 1 to call up the window shown in Figure 5 51 and choose CDNN txt from the drop down list under Save as type Specify a filename and location and click Save Computer amp 0s C cy HP_RECOVERY I ab 4 il File name in Save as type ProBinan ProBinaryA dsx ProBinary dsb Hide Folders ProDataStore dsa APL ASCI SPECTRAKINETIC glb APL ASCT kin JCAMP DX 5 0 jdx i CONN tet SimpleCSV csv ProDataCsV csv ProData TSW tect ol 140 aA e E ann nr Figure 5 51 the Save Selected Traces window Launch CDNN as previously described and open the file that you have just saved This example uses the lysozyme data described above Unlike the examples in the Sample Files folder these CD data are in millidegrees Care is needed to ensure that the results are meaningful Document 4207Q108C03 01 Page 116 of 133 Applied Ultrasensitive Spectr
3. Circular Dichroism mdeg 180 210 220 230 240 VVavelength nm Ready K Figure 2 10 CD spectrum of lysozyme raw data The program Pro Data viewer will automatically display the data as it is collected an example is given in Figure 2 10 Double clicking anywhere on the plot will autoscale the display When any CD spectrum is measured on Chirascan other properties are simultaneously recorded These include the voltage applied to the detector the temperature and the absorbance spectrum if selected on the Options panel They can be viewed by right clicking on the y axis variable 2 5 Viewing and manipulating spectra If you have just measured a CD spectrum Pro Data Viewer will be running and the CD spectrum will be displayed If Pro Data Viewer is not running launch it using the icon in the Pro Data SCP toolbar or if the Pro Data SCP is not running either from the desktop icon The program will open at the current working directory You can change the location of the working directory by selecting or creating a different folder and using the Set Working Directory option on the Directory menu Section 5 7 3 The working directory is where new spectra will be saved Document 4207Q108C03 01 Page 29 of 133 AppliedP hotophysics Ultrasensitive Spectroscopy for the Life Sciences EEE wena ee nye re Ome 5 aes Chirascan Series User Manual ze Pro Data Viewer ei x C Program Files x86 Applied Photoph
4. Sampling limits can be changed but the default values are best suited to routine operation The minimum time per point will be 25 us multiplied by 10 samples i e 250 us the maximum time per point will be 25 us multiplied by 4000000 samples i e 100 seconds These limits apply to all experiments whether set up using constant time per point or using adaptive sampling they are particularly useful in placing limits on the extremes of adaptive sampling 4 7 The Sequencer panel Sequencer Repeats 2 Triggered Spectrum T Kinetics Acquire Pause imed intervals z rael Timed intervals m Spectra Kinetics Figure 4 15 the Sequencer panel The Sequencer panel Figure 4 15 allows you to specify the type of measurement you will make and to control the starting stopping pausing and resuming of data acquisitions Tick the Repeats check box to run repeats of the same experiment and enter the number of repeats in the adjacent edit box The between individual measurements in Timed intervals which is the time between the beginnings of successive repeats if it is set to be shorter than the time for each run then each run will begin after the previous one All data from repeat runs will be written to a single Datastore The radio buttons allow you to select the type of measurement you will carry out It will usually be Spectrum for Chirascan Click Acquire to begin an acquisition according to the instructions in the SCP clicking Stop whi
5. Ultrasensitive Spectroscopy for the Life Sciences Sect AE E A ER 2 op Chirascan Series User Manual 2 8 4 Selecting the time per point The time per point is the time taken for data sampling at each step The longer the time taken the better the signal to noise ratio the selected time per point will therefore be a compromise between data quality and experimental duration In general the signal to nose ratio S N is proportional to the square root of the time per point thus doubling the time per point will reduce the noise level by a factor of V2 trebling the time per point will reduce the noise level by a factor by a factor of V3 and so on 2 8 5 Measuring protein spectra Measuring the CD spectra of proteins is perhaps the most common use to which the Chirascan is put The following paragraphs give some hints and tips on how to ensure that you your protein CD spectra are of the highest quality It is not possible to cover all contingencies but these guidelines should provide at least a starting point in helping you to find the optimal experimental conditions 1 Use a short pathlength if measuring into the far UV 2 Use a bandwidth lt 1 nm to ensure the detector is working within its linear regime especially as you go to the near UV where samples absorb less light With the sample in place the detector high voltage should not drop below 200 volts If it does reduce the bandwidth 3 If you wish to go below 180 nm consider usin
6. cccceeeccecseseeeeeseeeeecaeseeeeaeeeeeeseaeeeeseaueeessadeeesseeeeesaeeeeeseaeeeessaneesens 97 Figure 5 18 Options for exporting data ccecccccsescceceeeeeeceeeeeeeeeeeeeeseeeeeeeseeeeeeseeeeeeseaeeeeesegeeessaeeessageeeeseeeeeessegeeeeas 98 Figure 5 19 Stepped temperature ramp showing first Cell CD spectra cc eecccccccccseeeeeeeeeeeeeeaeeeseeeeeeeeesaaaeeeeeeees 98 Document 4207Q108C03 01 Page 17 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences reeeeseeeseceeceeeeceecees Chirascan Series User Manual Figure 5 20 the dimension MENU cccccceeeeececeeeceeceeeeeecaegeeeeaeeeeeesseeeesseeeeesaeeeesseeeeessaeeessegeeessaeeeeesseeeeessaeeeseas 99 Figure 5 21 CD spectrum for each of the four cells at 20 C snaannnnnnennnnnonennnnnsnnnnnnosrnrnnenrnrnnnnsrnrrrnsnnrrressrnrerennnne 99 Figure 5 22 Decompose Data dialog DOX ccccccccccccceneeeeeeeeeeeeeeaeeaeeeeeeeeeeaaeeaeeeeeeeeeesaaeaseeeeeeeeesaaaaseeeeeeeesaaaaenes 100 FIGURE 5 23 DECOMPOSE data dialog DOX siisii a T ine 100 Figure 5 24 the Decompose data dialog box for a 3 dimension Datastore cceccccccsseeeeeeeeeeeeeeeeeeeaeeeeeeeesaaees 100 Figu 5 25 the launchpad 100 Da sisena T E Ta E 101 Figure 5 26 the graphical display toolbar cccccccccsseececeeeeeeceeeeeeeeeeeeeeeaeeeeseaueeessaeeessegeeeeseaeeeessegeeessneeessageees 102 Figure 2 FAS Filo MoU weer cetersecrasch cect erinin aveansahentssussdodectista
7. Ultrasensitive Spectroscopy for the Life Sciences PEATE roe Chirascan Series User Manual The first three files are backgrounds the second set of three are the lysozyme spectra 1 Select the first group of three files and click Average 2 Select the second group of three files and click Average Two files named AVERAGE xxxxxxxxxx with identifying numbers taken from the computer timer will be added to the Visible Traces list and plotted in the display The first of these will be the average of the backgrounds and the second the average of the lysozyme spectra 3 Select the first AVERAGE file and click Set Background 4 Select the second AVERAGE file and click Subtract Background The file SUBTRACTED background subtracted spectrum will be added to the Visible traces list and plotted in the display Select the SUBTRACTED file and click Smooth The Smoothing dialog box will appear Figure 5 11 Strcottans Senay toion o iea joren o p m Size v surance Pe aca pli Size 7 aL seias a naman 13 564 3 E Subtracted 0 3 E Subtracted 0 8 713 E Smooth s 0 z 8 683 E Smooth s 0 E 3 862 E 3 840 E 1 003 E 5 839 E 5 846 5 10 689 10 689 180 00 196 00 21200 228 00 24400 260 00 180 00 196 00 21200 228 00 24400 260 00 Wavelength nm Wavelength nm 0 1390 0 4026 E Smooth r 0 E Smooth r 0 2 00493 2 01633 3 0 0405 3 0 0760
8. A E Figure 5 26 the graphical display toolbar Saves all the traces in the display into a new file Click on this icon to call up the Save As dialog box from where you can specify a filename and choose a location for the new file Prints the active document Causes an edit box to appear enabling you to record information in addition to that recorded automatically or entered through the Setup dialogue box in the SCP Calls up a history of the operations performed on the trace Print the active document Sets a selected trace as a background Clicking again will reset the trace to its original status Adds a previously defined background to a selected trace or traces It is useful if a background has been subtracted inadvertently from a trace This icon is inactive if no background has been defined Subtracts a previously defined background from a selected trace This icon is inactive if no background has been defined Opens the Curve fitting dialog box enabling one of a number of models to be fitted to a selected trace Section 5 3 2 Averages the selected traces the average will be added to the plot Averages all the traces in the display plots the average and removes the original data Opens the Simple math dialog box that enables you to add subtract multiply or divide a trace to from or by a constant Opens the Smoothing dialog box enabling a reversible Savitsky Golay smoothing operation to be performed on a select
9. A spectrum can be selected in a number of ways Once selected it changes colour to black is highlighted with selection marks and cross hairs appear on it The value of the x and y coordinates at the position of the cross hairs are shown above the graph Figure 5 4 Single spectrum selection methods are listed below e Place the mouse cursor close to the trace of interest Left click to select it Click a second time to deselect it e Right click anywhere in the display window to reveal a pop up menu Choose Select Specific Traces to open a dialog box containing a list of the spectra displayed Click on a filename to select it and OK to close the dialog box e On the Trace menu click Selection Dialog to open the Trace Manipulation dialog box Section 5 3 1 Click on a filename to select it and OK to close the dialog box e From the toolbar click on the icon to open the Selection dialog box Click on a filename to select it and OK to close the dialog box Document 4207Q108C03 01 Page 88 of 133 Applied hotop Ultrasensitive Spectroscopy for the Life Sciences Se ea eee AT Chirascan Series User Manual C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PLO004 dsa Circular D C B A File View Trace Math Scale Window Help gaai 0 0 S la A e Lyso 0_5mm PL0004 dsa v X 221 500000 Y 0 974260 Circular Dichroism mdeg 180 190 200 210 220
10. Absorbance Fuorescence Data Acquisition File Names Startup Preferences _ Auxiliary Measurements Default Options Temperature V AutoPM HY Figure 4 32 the Absorbance tab On the Absorbance tab Figure 4 32 preferences for absorbance measurements can be set In the Default Options panel select to measure spectra either at constant detector high voltage HV AutoPM not selected or at an HV Chirascan or emulated HV Chirascan plus that is automatically optimized at each point in the spectrum AutoPM selected Constant HV or emulated HV will normally give the more accurate absorbance measurement because there is no risk of hysteresis associated with sharp changes in the detector HV or HV but you must be careful to ensure that the detector is not saturated during the measurement Selecting AutoPM will remove that possibility and because there are unlikely to be big changes in HV or emulated HV from one point to the next using AutoPM will not degrade the absorbance measurement in any significant way It is recommended that this option is selected 4 14 4 The Fluorescence tab Preferences ewer CD Absorbance Fluorescence Data Acquisition File Names Startup Preferences Auxiliary Measurements Temperature HY Figure 4 33 the Fluorescence tab There are no active panels on the Fluorescence tab Figure 4 33 Document 4207Q108C03 01 Page 80 of 133 Applied hotophysics U
11. AutoPM Because of this process the detector gain will routinely vary during a scan and between reference background and sample CD measurements so that the conditions for the normal absorbance calculation are not met In order for spectrometer to deliver accurate absorbance spectra the following method has been developed which accounts for the variable gain element in the calculation For the LAAPD detector the internal gain of the detector is constant while an amplifier stage is used to control the absolute gain The absolute gain and thus the change in gain between two measurements is therefore always known This is unlike the PMT detector where the gain has to be estimated For the Chirascan PMT the current gain is given by Equation 6 7 2 n La a a kn Te n 1 6 7 u where u is the current gain Z4 and Ic are the anode and cathode currents respectively n is the number of dynodes in operation k and a are constants dependent on photomultiplier design and material the collection efficiency and HV the applied high voltage Document 4207Q108C03 01 Page 126 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PPS rh eee ae Chirascan Series User Manual As described above during a scan a constant DC signal voltage is targeted by varying the current gain of the photomultiplier This is achieved by software control of the detector high voltage HV From Equation 6 6 the reference and sample DC si
12. Cancel Help Reset Fark kona Figure 4 27 the Monochromator window Chirascan Series User Manual In the Wavelength panel the slider bar or the edit box can be used to specify a wavelength clicking the Set button will drive the monochromator to the chosen wavelength Likewise the Bandwidth panel can be used to set the bandwidth These settings are the ones to which the monochromator will drive when the software is started The Status panel reports what the monochromator is doing when it is executing your instruction the status reported would be Updating and No respectively in the Current action and Reached target boxes and on completion the text would read Ready and Yes as shown above The temperature of the mono block is recorded and can be refreshed using the button adjacent to the reported temperature Reset causes the mono to verify its limits both in wavelength and bandwidth Park mono puts it into a safe position ready for transportation When you are happy that the parameters are correctly set click OK to close the panel without implementing the changes click Cancel The Calibration File panel should be used only under instruction from APL technical support Document 4207Q108C03 01 Page 75 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences EE S eee 2 Chirascan Series User Manual 4 13 3 The attenuator A the Attenuator icon displays the Attenuator Shutter Control wi
13. Detector 1 CD detector Detector 2 fluorescence detector Figure 6 2 schematic of the spectrometer optical train The standard light source is a 150 watt ozone producing xenon arc lamp although other options are also available Section 3 4 An optimised ellipsoidal focussing mirror M1 ensures maximum efficiency and light capture The lamp housing accommodates a pre aligned lamp assembly for easy lamp replacement The monochromator features a dual polarising prism design This yields pure linearly polarised monochromatic light and allows high spectral bandwidths in the far UV The digital drive system uses a stepper driven cam with digital calibration and active temperature compensation Three coupled high precision slits are driven by a second stepper motor and a third is used to control a variable aperture shutter The entire monochromator assembly is sealed with strategically placed nitrogen purge inlets and outlets This enables the interior atmosphere to be replaced with nitrogen rapidly and effectively and also allows the nitrogen atmosphere to be retained for long periods after use F 7 optical coupling is maintained throughout the optical path This has been chosen to maximize light throughput without compromising focussing performance The linearly polarised monochromatic beam that emerges from the exit slit of the monochromator is then re focussed down through the photo elastic modulator PEM This device which acts as a dyna
14. Export Data Decompose Data Temperature Ramp Data Delete Rename Properties Figure 2 19 File manipulation pop up menu Document 4207Q108C03 01 Page 35 of 133 AppliedPhotophysix Ultrasensitive Spectroscopy for the Life Sciences Sect E AE A OER EA Chirascan Series User Manual Select Export data to bring up the dropdown list in Figure 2 20 and choose an appropriate format for export to another program Select Data Format Probinarys ProBinary Proll ataS tore APL ASCII JOSMP D 5 0 Simples ProD atals Figure 2 20 options for exporting data CSV format is suitable for third party programs such as Excel other options relate to formats derived from other APL products 2 6 3 Converting data for use with third party programs HEN This icon is installed on your desktop and will start the APL data converter Figure 2 21 APLDatato Fig APL Pro Data Converter es eee File View Help Drag and Drop DataStore files 4b here For Help press Fl Figure 2 21 the APLDataConverter target The APLDataConverter program can be used to convert multiple files to other formats Start the program click the icon to open the dialog box shown in Figure 2 22 set the required options and close the dialog box Select the files in their Windows folder and drag them onto the APLDataConverter target The created files will be saved in the same folder as the originals Document 4207Q108C03 01 P
15. Figure 4 3 the Live Signal display cnancesscscatesrcadaboareisstantenauiusiawstentensuibendariaboudiantiesientsiwntinduibevidedetveidaidiertensaiastsaaulestes 59 Figure 4 4 the Signal panel with Circular Dichroism selected cccccseeeeeceeeeeeeeeeeeeeeaeeeeesseeeeesaeeeeseeeeesaeeeeeas 60 Figure 4 5 the Quickview CD background dialog DOX cccccccccccceeeeeceeseeessssseeeeceeeeeeeeeeeessessaaeseseseseeeeeeeeeeeeess 61 Figure 4 6 the Signal panel with Absorbance SeleCte cccceccccccseseeecaeeeeeseneeeeeeuseeeceeeeeesseeeeessaeeeeseeseeessneeseas 62 Document 4207Q108C03 01 Page 16 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences pRSanes sReatenerrsetenenes Chirascan Series User Manual Figure 4 7 the Signal panel with Voltage selected ccceecccccsseeeeeeeeeeeeeeeeeeeeueeeeeseaeeeeseeueeesseeeeesageeeeseaeeeesseneeseas 63 Figure 4 8 the Shutter Attenuator Dane ll ccccccccsseeeceeeceeeeceeeeaeeeeeeesseeeeeeeesueasceeeeseeesceeeeseeeeeeessaaeeeesessaaeeeees 63 Figure 4 9 the Monochromator Panel cccccccccseseecceeeeeeeaeeeeeeseeeeeeseaeeeessaueeeeseeeeeseaseeessaeeesseeeeessageeeessaeeeesseneeeeas 64 Figure 4 10 the Monochromator Scan Setup Advanced dialog DOX ccccecccccseeeeeeeeeeeeeeeeeesaeeeeeeeeeaaeeeeeessaaeeeees 65 Figure 4 11 the Trigger and Timebase Panels cccccccssececcseeeceeceeeeeecaeeeeeeneeceeseeseeesseueeesseneeessaeeeeese
16. but which can be dynamically adapted by the algorithm to improve the scan efficiency as the light throughput varies These values are the default sample size 20000 or 0 5 seconds time per point and an HV of 800 volts If adaptive sampling is not active and the scan proceeds a 20000 point average will be calculated at every wavelength in the scan range If adaptive sampling is selected then the sample size and therefore the sample time is increased or reduced according to the relative sample size function Y exp 0 003 800 HV 6 22 Thus at the reference HV of 800 volts the adaptive sampling scaling factor Y is 1 0 at an HV of 500 volts the sampling scaling factor is 0 407 and at an HV of 1000 volts the scaling factor is 1 82 So at 1000 volts there would be roughly twice as much time spent acquiring each point as compared to the default time per point At HV levels of 500 volts each point would be measured in roughly 40 of the default time The net effect for a typical sample is that a scan should be faster with adaptive sampling than without for the same default sample size particularly if the scan extends into high light throughput regions of the spectrum This will be offset to some extent by increased time spent where light is limited in the far UV and in the presence of absorbing solvents but here there will be a small enhancement to the signal to noise It is also possible for the user to change the default numbe
17. c cceeeeeeeeeeeeees 116 Figure 5 51 the Save Selected Traces window ccccccsssssscceeceeeeeeseseeeeeseeseeeeseeeeeeeeseseaseeeeeeesesaeaaseeeeseeenaaaaages 116 Figure 5 52 the CD Signal Type WINGOW ccccccccccseeeeeeeeeaeeeeeeeesaeeeeeeeeseeeeeeeeseeaseeeesseeaeeeeeesaaaseeeessaaeeeeeesaaess 117 Figure 5 53 the Deconvolution WINKOW ccccssececcseseecceaseeecceusceecsugeeecsaeeecsaseeessaseeecsaeeesseuseeessaueeessaseesssaaeees 117 Figure 5 54 the CD Signal Type dialog box for an UnCcharacteriSed protein ccccccesseeeeeeeeseeeeeeeeesaeeeeeeeesaeees 118 Figure 6 1 Schematic of the relationship between the Chirascan subsystems ccceeeeeeeeeeeeeeeeeeeeeeeeaaeees 119 Figure 6 2 schematic of the spectrometer optical train cccceecccecseeeeecseeeeeeeeeseeeeeeeeeesaeeeeesseeeeseaueeessaneeesnaasess 120 Figure 6 3 quantum efficiency of the LAAPD and PMT detectors ccccccccsesceeeeeeeeeeneeeeeeseeeeeeeeaeeeesaaeeeeenaaeees 122 Figure 6 4 LAAPD amplifier gain and emulated HV ValUC cccccceccccceceseeseeeeeseeeeeeeeeseeeseeeessaeeueeeeesaaeeeeesaaaees 122 Figure 6 5 noise variance against HV at constant DC target ccccccecccseeeeeeeeeeeeeeeeeeeaeeeeeeeeeaeeeeeeeesaaeeeeeeesaaees 130 Figure 6 6 relative sample size as a function Of HV 1 0 ceceececeeceeesseeeeseeesseeeesseauseeeesseaaseeeesseaaeeeenseaseeeeensagees 131 Figure 6 7 raw smoothed and residual
18. lt 4 nm 200 nm 0 1 nm lt SBW lt 6 nm Table 2 2 maximum bandwidth at low wavelengths The selected bandwidth is therefore a compromise between data quality and spectral resolution Typical values are samples in solution 0 5 nm lt SBW lt 2 nm gas or vapour samples 0 1 nm lt SBW lt 0 5 nm Specific examples are given in Section 2 9 2 8 3 Selecting pathlength and concentration The pathlength and concentration of a sample should be chosen to optimize the light throughput to the detector If the absorbance is too high the light throughput will be too low for the detector to measure accurately if the absorbance is too low the detector will not be able to resolve the difference between the left and right circularly polarized signals accurately The optimal value for the absorbance when making CD measurements is about 0 87 absorbance units AU 14 light throughput About 2 5 AU is usually the realistic upper limit for the absorbance ideally it should not exceed about 2 AU It is good practice to run a simultaneous absorbance scan when performing a CD experiment in order to verify that the absorbance is within the working range The absorbance can be adjusted by changing the concentration or pathlength following the Beer Lambert law Axc l Where A is the absorbance c is the concentration and is the pathlength the proportionality constant depends on the sample Document 4207Q108C03 01 Page 38 of 133 Applied
19. unknown typical settings are samples in solution 0 2nms SSsinm gas or vapour samples 0 1 nm lt SS lt 0 5 nm Examples are given in Section 2 9 Note that the analysis of protein spectra using the public domain software CDNN requires a 1 nm step size Document 4207Q108C03 01 Page 37 of 133 AppliedPhotoph Ultrasensitive Spectroscopy for the Life Sciences Spc E AE A ER AA Chirascan Series User Manual 2 8 2 Selecting Spectral Bandwidth SBW The spectral bandwidth should be selected to maximise the light reaching the sample without overly compromising the spectral resolution In general the signal to noise ratio S N is proportional to the square root of the light throughput and the light throughput is proportional to the square of the bandwidth Consequently S N is linearly proportional to the bandwidth Doubling the bandwidth will reduce the noise level by a factor of 2 trebling the bandwidth will reduce the noise level by a factor of 3 and so on However a wider bandwidth may result in some of the finer details of the spectrum being obscured and there is limit to the maximum bandwidth that can be set This varies with wavelength because of the non linear dispersion of light by prisms dispersion increases and therefore maximum bandwidth decreases with decreasing wavelength The SBW ranges for the shorter wavelengths are given in Table 2 2 Wavelength SBW range 170 nm 0 1 nm lt SBW lt 3 nm 180 nm 0 1 nm lt SBW
20. 4 Zooming and re scaling the display using the mouse To zoom in to a particular part of the display click and hold the left mouse button while moving the pointer to create a box around the area of interest On releasing the left mouse button the newly created box will expand to fill the display window Figure 5 5 To return to full display double click in the display area Document 4207Q108C03 01 Page 89 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences cen eecescccccecccccscsccenccesccccccoscees Chirascan Series User Manual EB cAProgram File 86 Applied Photophysics Chirascan Example Lysozyme Spectra Lyzo 0_5mm PL0004 dza Circular D S EF CAProgram Files xB6jVAppiied Photophysics Chirascan Examples Lysozyme Spectra Lyzo 0_Smm PLOOOS dza Circular D gt E fie View Ixe Math Scale Window Help lix E File View Trace Math Scale Window Help S0fw Hi E ME jesas i G i c BD CHD om Circular Dichroism mdeg Circular Dichroism mdeg i ih EESO eee ee eee Se Cae Seen ee ee Spee See Het ae 180 190 200 210 220 230 240 250 260 E 190 195 200 205 210 215 220 225 230 VVavelength nm VVavelength nm Figure 5 5 full display left and zoom using the left mouse button right Use the mouse wheel to expand or contract both axes simultaneously Hold down the Ctrl key and use the mouse wheel to expand or contract only the horizontal
21. AU or at most 2 5 AU reasonable quality spectra should be obtainable 8 Remember that light intensity at low wavelengths may cause photodegradation of the protein Keep the bandwidth and the time per point as low as possible commensurate with obtaining reasonable quality data If there is any doubt repeat the experiment to ensure that the results are reproducible or perform a preliminary experiment to establish the timescale over which photodegradation occurs The recommended conditions are summarized in Table 2 3 Lower wavelength limit Solvent Pathlength Protein concentration H2O dilute buffer or DzO 0 2 0 5 mg m H2O or DO 0 5 1 0 mg ml 1 0 mg ml Table 2 3 recommended conditions for measuring protein CD spectra Document 4207Q108C03 01 Page 39 of 133 AppliedPhotophysix Ultrasensitive Spectroscopy for the Life Sciences Sect ever AE A OER EA Chirascan Series User Manual 2 9 Example spectra There are several files installed on the hard disk that can be used as templates to set up the Pro Data interface or as tools to familiarise oneself with the use of Pro Data viewer The files are held in C Program Files Applied Photophysics Chirascan Examples and can be accessed using the icon from the Pro Data interface toolbar and in Pro Data viewer by navigating to the directory in the launchpad 2 9 1 Alcohol dehydrogenase The CD spectrum of alcohol dehydrogenase plotted as CD mdeg against wavelength nm is sh
22. Background CD spectrum An example of a background CD spectrum is shown in Figure 2 8 You do not need to use the default name for sh the background you can specify a seed name by clicking on the File Names icon in the toolbar of the Pro Data interface or selecting Configuration gt Preferences gt File Names Section 4 14 6 and specifying seed names for the backgrounds and spectra Figure 2 9 File Names File Names Spectrum Protein Kinetics Kinetics Spectra Kinetics Proetine SK Baseline nitrogen backgorund PDA Pda 4 gt 4 gt 4 gt 4 gt 4 gt 4 gt PDA Baseline Pdab Figure 2 9 the File Names dialog box 2 4 2 The CD spectrum Once you have measured the CD background replace the blank with your sample As the conditions for the measurement of the sample will be identical to those for the blank go straight to the Spectrum panel of the SPC and click Acquire The CD spectrum will be recorded under the conditions applied to the blank and stored as a file called Spectrumxxxx dsx unless you have specified a different seed name Section 4 14 6 Document 4207Q108C03 01 Page 28 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences Se EE eee Chirascan Series User Manual 7 C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0007 dsa Circular D eale ea A File View Trace Math Scale Window Help d amp g EEE x
23. C 2 edition Cambridge University Press 1992 Document 4207Q108C03 01 Page 133 of 133
24. Data Viewer File View Directory Help Windows Turret 4 CSA Single Window C Program Files x86 4 4L Go On line E at ee we i Allow Cary Files Figure 5 30 the Preferences menu The Preferences menu Figure 5 30 is used to set the conditions for the graphics display and on line status e Single Window when selected means that whenever a new graphical display is opened during data acquisition all previous ones are closed If it is not selected then whenever a new measurement is made the previous displays are not closed but will remain open on separate tabs If you are running an experiment that involves a lot of repeat measurements it is recommended that Single Window be selected if not you could end up with a large number of displays to close Single Window has no effect when opening files from the launchpad e Go On line will display the Connect dialog box Figure 5 31 on which you will be asked to supply the Chirascan IP Address It is used when Pro Data Viewer has been started as a stand alone program and you want subsequenily to establish a link with the spectrometer control program SCP If the two programs are running on the same computer select Local in the Connect window The IP address under these circumstances is always the default 127 0 0 1 Document 4207Q108C03 01 Page 104 of 133 Applied hotophysi Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual Spectrometer Control
25. Datastore Decompose Data Name Range Min Range Max Primary Wavelength 240 000000 340 000000 Cell 1 000000 4 000000 Temperature 20 000000 30 000000 Figure 5 24 the Decompose data dialog box for a 3 dimension Datastore Document 4207Q108C03 01 Page 100 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PPS rr he aE aE Chirascan Series User Manual The number of Primary boxes ticked will determine the lowest dimension Datastore created In the above example if all three Primary boxes were ticked a single three dimension Datastore would have been created with the range restrictions specified in Range Min and Range Max fields If two Primary boxes were ticked for example wavelength and temperature a single three dimension Datastore would have been created along with a two dimension file for each cell If only one Primary box was checked for example wavelength a single three dimension file would be created along with two sets of two dimension Datastores with wavelength temperature and wavelength cell as the dimensions each of which would be decomposed into the corresponding one dimension Datastores 5 6 Pro Data Viewer toolbar icons 5 6 1 Launchpad toolbar icons The launchpad toolbar is shown in Figure 5 25 Positioning the cursor over the icon will call up a brief description a fuller description is also provided in the status bar at the foot of the launchpad fae Pro Data Viewer File View Direc
26. EEE ae Chirascan Series User Manual 4 14 7 The Startup Preferences tab Preferences Viewer CD Absorbance Fluorescence Data Acquisition File Names Startup Preferences Emulation Start in Emulator Mode by default Startup Control Panel Mode Select the default Control Pane Mode Spectrum Kinetics Spectra Kinetics Figure 4 36 the Startup Preferences tab On the Startup Preferences tab the SCP can be set to open in Spectrum Kinetics or Spectra Kinetics modes For Chirascan the default Spectrum is usual You can also choose to start the software in emulator mode i e in the absence of any hardware but that is unlikely to be of interest during normal use 4 15 Recalibrating the Chirascan 4 15 1 Recalibrating the CD Note that you will need system administrator privileges to change the CD calibration factor 1S 10 camphorsulphonic acid 1S CSA is widely used for the calibration of CD instruments Some care needs to be taken in its preparation for it to be used for calibration purposes because it is hygroscopic and therefore difficult to weigh accurately However CSA has been studied in depth and is known to have an ellipticity of 335 millidegrees when a solution of precisely 1 mg ml is measured in a 10 mm pathlength cuvette It is also known that the extinction coefficient of anhydrous CSA is 34 5 at its absorbance maximum 285 nm and the concentration of a solution made up to approximatel
27. Figure 5 44 0 5 pb 2 epee nn en ten te nn te ee en nen en nnn een penne nee nee Circular Dichroism mdeg ras Circular Dichroism mdeg 180 190 200 210 220 230 240 250 260 460 190 200 210 220 230 240 250 260 VVavelength nm VVavelength nm Figure 5 44 single snoothed background corrected residuals left and their average right Document 4207Q108C03 01 Page 112 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences cies E A E ASOD Tete Chirascan Series User Manual 5 10 Using CDNN to calculate the secondary structure of proteins CDNN is a third party program that is included with Pro Data Viewer by permission of its author to assist users in calculating protein secondary structures Any publication of results calculated using CDNN should refer to the author see the CDNN manual on the installation disk for details Details of the program and its operation are given in the CDNN Help system and the user is advised to use those guidelines in conjunction with those given here 5 10 1 Getting started Launch the CDNN software using the l box Figure 5 45 File Edit Deconvolute Options Help Figure 5 45 the Simple Spectra dialog box From the File menu select Open On first use the program will open at the Sample Files folder Figure 5 46 C Program Files Applied Photophysics CDNN Sample Files Open File Look in F Sample Files H gtochrom_e H
28. File Directory Preferences Help Wr Close All Viewer Windows Figure 5 28 the View menu On the View menu Figure 5 28 Close all viewer windows is a quick means of closing all Pro Data Viewer graphical displays It is useful if Single Window in the Preferences drop down menu has not been selected e Always on top is a toggle to force Pro Data Viewer to be the top window Document 4207Q108C03 01 Page 103 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences AIET 2 Chirascan Series User Manual 5 7 3 The Directory menu eal Pro Data Viewer File View Directory Preferences Help Windows Mowe Root Directory Up One Lewel C Program Fi Set Working Directory E a E E Mew Folder j i Browse for Folders Figure 5 29 the Directory menu Back to Working Directory The Directory menu Figure 5 29 can be used to set the current working directory or to return to it e Move Root Directory Up One Level will take you back through the directory tree one node at a time e Set working directory will define the currently selected directory folder as the working directory e Back to working directory will take you directly back to the current working directory e New folder will create a new folder in the currently selected directory e Browse for folders allows you to browse easily to a folder that can then be set as the working folder 5 7 4 The Preferences window g9 Pro
29. History shows a history of the operations performed on the file for example background subtraction smoothing and fitting Document 4207Q108C03 01 Page 106 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual 5 8 3 The Trace menu iles x86 Applied Photophysics Chirascan Examples Lysozyme Sp Math Scale Window Help i so Show Trace Name 1 Selection Dialog D Select All Ctrl Spc Deselect All Alt Spc LO Remove Selected Del f77777 niai Remove Unselected Traces Shift Del Toggle Selected as Baseline E r Add Baseline Subtract Baseline Remove Overlaid Traces Shift R Recompute Trace Colours F10 Reset Shift Enter Figure 5 36 the Trace menu On the Trace menu Figure 5 36 Show Trace Name is a toggle to show hide the name of a highlighted trace when the mouse pointer is placed near to it Selection Dialog calls up the Trace Manipulation dialog box Section 5 3 1 Select All selects every trace in the display Deselect All deselects every trace in the display Remove Selected removes from the display any currently selected trace Removed traces can be recovered using the Trace Manipulation dialog box Section 5 3 1 Remove Others removes from the display any trace that is not selected Removed traces can be recovered using the Trace Manipulation dialog box Section 5 3 1 Toggle Selected as Baseline sets unsets a selected trace as a base
30. MANUAL MAY RESULT IN A HAZARDOUS SITUATION AND WILL VOID THE MANUFACTURERS WARRANTY The Chirascan and Chirascan plus spectrometers are powered by the mains electricity supply which can produce an electric shock leading to serious injury or death Do not connect or disconnect the spectrometer from the mains supply unless the supply is powered off at source Ensure all communications and electrical connections are made before powering on the spectrometer Exercise care during operation and do not operate units with their covers removed Operate the spectrometer using only the cables provided Never operate a spectrometer with damaged cables The metal components of the spectrometer can produce an electric shock leading to serious injury or death if they are not earthed grounded The design of the spectrometer provides protection against electric shock by earthing appropriate metal components This protection will be lost unless the power cable is connected to a properly earthed outlet It is the user s responsibility to ensure that a proper earth connection can be made The photomultiplier tube PMT detector used with the Chirascan and the avalanche photodiode APD detector supplied with the Chirascan plus operate at high voltages and can produce an electric shock leading to serious injury or death Do not connect or disconnect the detector from the spectrometer unless the spectrometer is powered off lamp that produces intense ultraviolet radia
31. Monochromator Scan Setup Advanced dialog box Figure 4 10 and click on Save As Default Click OK to take you back to the Pro Data interface Document 4207Q108C03 01 Page 64 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PP re es eee ae Chirascan Series User Manual Monochromator Scan Setup Advanced Single wavelength Wavelength list Index Wavelength 280 0 279 0 278 0 277 0 Stat 280 0 Stop 200 0 Step 1 0 aS a e a B none whH CO Number of entries in list 94 Append to list o Clear List Generate List gt gt Bandwidth nm Save As Default Cancel Figure 4 10 the Monochromator Scan Setup Advanced dialog box 4 4 2 Advanced mono setup discrete wavelengths The Monochromator Scan Setup Advanced dialog box Figure 3 10 can be used to set the software up to perform single wavelength scans Such measurements are useful when following a change in property as a function of some independent variable other than wavelength temperature for example Select Single wavelength and specify the wavelength in the associated edit box Click OK The dialog box will close and when you click on the Start button in the Spectrum panel of the interface the monochromator will drive to the selected wavelength and measure the CD or other chosen property of the sample at that wavelength It is also possible to create a list of discrete wavelengths Clear the wavel
32. Replace original data with smoothed g 8 713 3 862 0 988 5 839 Circular Dichroism mdeg 10 689 180 00 212 00 228 00 Wavelength nm 0 0493 Residuals 0 0405 0 1303 180 00 212 00 228 00 Wavelength nm Tance E Subtracted 0 E Smooth s 0 E Smooth r 0 Chirascan Series User Manual P Smoothing Savitsky Golay Window Size Circular Dichroism mdeg V Save Residual On Graph Replace original data with smoothed 212 00 228 00 Wavelength nm 244 00 260 00 0 1633 0 0760 Residuals 0 3153 180 00 212 00 228 00 Wavelength nm 244 00 260 00 E Subtracted 0 E Smooth s 0 E Smooth r 0 Figure 2 15 an appropriate smooth left and an inappropriate one right Enter the number of degrees of smoothing required in the Window Size box This value will depend on the step resolution used during the measurement and the sharpness of the features in the spectrum The number should be as large as is possible without causing distortion of the spectrum Examination of the residual plot will show if any distortion has occurred If the noise is randomly distributed about zero the smoothing process has not caused any distortion and importantly you have the evidence to prove it An example of an appropriate smooth in which 5 degrees of smoothing were used and an inappropriate one in wh
33. a hazardous situation which if not avoided could result in minor or moderate injury The sign to the left is used to indicate a hazardous situation which if not avoided could result in death or serious injury OTHER INFORMATORY INDICATORS USED IN THIS DOCUMENT leya 4 The sign to the left is used to indicate a situation which if not avoided could result in damage to the instrument HAZARD INDICATORS USED ON THE SPECTROMETER OR ACCESSORIES Note that these hazard indicators may be either coloured as below or as black and white The sign to the left is a general hazard indicator indicating the presence of a hazard that is either described by text accompanying the sign or in this User Manual The sign to the left is a High Voltage hazard indicator Document 4207Q108C03 01 Page 5 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual ESSENTIAL SAFETY INFORMATION MAKE SURE THAT YOU HAVE READ AND UNDERSTAND ALL THE SAFETY INFORMATION CONTAINED IN THIS DOCUMENT BEFORE ATTEMPTING TO OPERATE THE CHIRASCAN SERIES SPECTROMETER IF YOU HAVE ANY QUESTIONS REGARDING THE OPERATION OF YOUR SPECTROMETER PLEASE CONTACT APL TECHNICAL SUPPORT SECTION AT THE ADDRESS SHOWN ON THE FIRST PAGE OF THIS DOCUMENT OBSERVE ALL SAFETY LABELS AND NEVER ERASE OR REMOVE SAFETY LABELS PERFORMANCE OF INSTALLATION OPERATION OR MAINTENANCE PROCEDURES OTHER THAN THOSE DESCRIBED IN THIS USER
34. accurate if the calibration value is correct In the file autoabs conf stored in C Program files Applied Photophysics Chirascan Config you will find a single number This is the scaling factor that relates the true absorbance to the derived absorbance To adjust this scaling factor you must measure the true Document 4207Q108C03 01 Page 83 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual absorbance of a sample and then its derived absorbance in CD mode Accurate knowledge of the concentration of the sample is not required Calculate the correction factor from Factor True absorbance Derived absorbance and multiply the scaling factor in the autoabs contf file by the correction factor A didymium absorbance calibration standard with an absorbance of about 0 5 AU is used for the initial calibration by APL production department and gives good results but any absorbing sample will do For example a 3 mg ml solution of 1S CSA in a 10 mm pathlength cell should give an absorbance of approximately 0 45 AU relative to water at its absorbance maximum of 285 nm It is not essential to use CSA and you may choose to use another compound all that is important is that the sample has a significant absorbance at a convenient wavelength For 1S CSA set the monochromator range to 270 nm to 300 nm the step size to 0 5 nm and the bandwidth to 1 nm Set the time per point to 1 second for othe
35. axis Hold down the Shift key and use the mouse wheel to expand or contract only the vertical axis Figure 5 5 EJ CAProgram Files 86 VApplied NCh mgle irona Spectrellyo 0 5mm POCO dae Croslar DL lh EJ CAProgram Files x86 Appied Photophysics Chrascan Examples Lyraryme Spectra kyso 0 Saum PLODOL dre Circular D H E Citrogram Fes x86 Apphed Photophysicy Cheascan Examples tysoxyme Spectralyso 0 Sm PLOOOA cea Circular D H Ei Fle Vew Trace Math Scale Window Help s e Jeja Ti pie yew Jace Math Scale Window Help alx E Ble yew Trace Math Scale Window Help x g S608w E 00mm gsnaw 8 9 e gsang 38 1 OD OH 8 BD Circular Dichroism mdeg Circular Dichroism mdeq Circular Dichroism mdeg 2 170 180 190 200 210 20 230 x0 20 260 270 180 180 200 210 220 230 240 250 260 VVavelength nm VVavelenath nm VVavelength nm Figure 5 6 zoom using wheel alone left Ctrl wheel centre Shift wheel right The zoom functions can be used in any combination To return to the default full screen display doublet click at any time in the display area 5 2 5 Displaying different properties Pro Data Viewer always plots the dimension on the x axis and the selected property on the y axis Right clicking the y axis legend will display a menu of the properties recorded during a measurement Figure 5 7 To change the property displayed select one from the list by double clicking it Y Axis Options Cir
36. eea E cn ane ada E E E E edie ane Gineanneishiavenanes 49 3 1 The spectrometer layout sects caitaianiainatetidawsinnetinnnamedcrndensantnmaiandanusanealicnxaaedcauiuauesnentatdawaiamsdlvancessnaaeiuaassunchdiedanntis 49 32 NSC I aN connections we sheet cteestico sc acteustintiicedanecapeoinwesdinsneacesetatieddaniaiusssrinvetdartenasieersuetiicrsauasineinesieetaaussrratietien 49 FELN UVSC On CHOI S e EE E EEE A E E EEA E EEE E EO 49 3 2 2 Mod le CONNECTIONS sinisesse an rran E E AS S 50 Document 4207Q108C03 01 Page 12 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences pRSanes sReatenerrsetenenes Chirascan Series User Manual 3 3 Nitrogen purge GAS CONNECTIONS ccccsceccccseeeceeseeeeeeceeeeeeeneeeeeeseeeeeeseaueeessaeeesseeeeessaueeessaeeessageessseeeeeessess 51 iP IGM O E E ameveryeieadunsreianlnmanais emai iaiateaiais cesarean venanipeetannars 51 ET EEC g 7 eee eee E eee eee ee ee ee ee 51 34 2 Standard xenon UN erreen TE aE ossueaneaadentandieouieedias 52 3 4 3 Mercury XENON IMO enr E E ONE E EET E S 52 34A Ozone dree Xenon AVIV anisses Ea r DEEE EDE 52 3 4 5 Lamp removal and replacement s sssscsisrresrecsrissrsesrisrrriseriresisisestrs sti torrir nn Eneak SENEE EKEN EN SAE oraa sasa Sa 53 3 5 The Spectrometer Sample Handling Unit ESHU cee cecccccecceeeeeseeeeeeeeeeseeeeeeeeeeeesseseeeeeseeeeeesssaaeeees 54 EDOL CONS eren E E E E 55 DOD CMEC TOO ltl ho serer veces aa RE EE A EEEE EANES 55 3
37. for subsets of the original data and the subsets saved as new files The process is recursive so higher dimension Datastores can be reduced to their simplest one dimension forms if required Below is an example of how to mine a two dimension Datastore Right click on a two dimension Datastore in the launchpad file list and select Decompose Data from the menu A Decompose data dialog box will appear Figure 5 22 Name Range Min Range Max Primary Wavelength 240 000000 340 000000 Temperature 20 000000 60 000000 Figure 5 22 Decompose Data dialog box In the example the dialog box shows that the Datastore has two dimensions wavelength and temperature and the ranges those dimensions span To reduce the range of temperatures to 25 C to 50 C and retain wavelength as the only dimension input these values into the temperature range boxes and clear the temperature Primary check box Figure 5 23 Decompose Data Name Range Min Range Max Primary Wavelength 240 000000 340 000000 Temperature 25 000000 50 000000 C Figure 5 23 Decompose data dialog box A new one dimension Datastore will be created containing spectra for every temperature from 25 C to 50 C Click Save as name the Datastore and refresh the launchpad directory tree The Datastore will be in a new folder created in the original folder Three dimension Datastores can be decomposed in the same way Figure 5 24 shows the Decompose data dialog box for a three dimension
38. from the CD spectrum of the sample To measure the CD background you must measure the CD spectrum of a blank The blank must be identical in every way to the sample that you want to measure except that the sample itself is absent For example if you intend to measure an aqueous solution of lysozyme in a 0 2 mm pathlength rectangular cuvette then the CD background is determined using the same cuvette containing only water Signal Circular Dichroism 15 1251 Imdeg Options 5 delta Absorbance milidegrees Fluorescence AutoPM Baseline Auto Subtraction Figure 2 5 the signal panel in the Pro Data interface On the Signal panel Figure 2 5 there is a drop down menu that contains the main data collection modes select Circular Dichroism In Options select either delta A delta absorbance or millidegrees as the units in of CD Select Absorbance if you wish make a simultaneous measurement of the absorbance spectrum of the sample Circular Dichroism millidegrees and Absorbance are selected by default Monochramator Wavelength nm 260 0 59 0 100 0 150 0 200 0 250 0 309 0 Bandwidth nm 1 0 Pe ea ere mee ee Aw eee fp eT ranli g Sa bow 2000 2 High 2000 E step 1 0 E sa Adv Figure 2 6 Monochromator panel in the Pro Data interface Set the lower and upper wavelength limits of the scan range for your experiment on the Monochromator panel of the Pro Data interface Figure 2 6 these will normally be th
39. initial starting parameters for the fit click the Estimate button to automatically generate a set of initial parameters which may not be very accurate The curve associated with these parameters is plotted in the preview window to the right To initiate the fitting of the parameters to the data click Fit The algorithm will iterate until convergence or the maximum number of cycles is reached If the fitting is successful the parameters will be updated and the calculated curve plotted in the preview window The associated residual trace the difference between the observed and calculated data will be displayed in the Residuals part of the preview window The errors associated with the calculated parameters are displayed next to the calculated parameters You will be able to judge the quality of the fit by inspecting the residual trace and the parameter errors If the errors are large compared with the parameters then it is likely that you have specified a fitting function which is more complicated than is justified by the data i e the result is under determined The number of variable parameters can be reduced by locking any parameter using the check boxes adjacent to the parameters but such locking must be justified and should be applied with care lf the fitting process does not converge within the selected maximum number of iterations a warning is given You may wish to increase the number of iterations up to a maximum of 1000 Be aware that 10
40. of the sample is obviously a useful measurement in its own right and is a useful supplement to the CD spectrum As a general rule optimal CD measurements are obtained when the sample absorbance in the cell is between about 0 8 and 1 0 AU 2 If the absorbance is too low the CD signal will be weak if it is too high insufficient light will reach the detector The absorbance spectrum also provides a measurement of the sample concentration and indeed can be used to calculate sample concentration given the observation cell pathlength This will enable accurate molar ellipticity and molar extinction values to be calculated An important ratio Known as the g factor or disymmetry factor which is the ratio of the CD to the absorbance can also be obtained AA Ae z A o 6 3 AA is the measured CD A the absorbance measured under the same conditions Aes is the molar CD and the molar extinction coefficient i e A Ae c l 6 4 A As C l 6 5 Document 4207Q108C03 01 Page 125 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences A Se eee Os Chirascan Series User Manual Where is the cell pathlength in cm and c the sample concentration in mol dm The g factor is a particularly useful quantity when estimating or assessing enantiomeric excess 4 6 3 2 Absorbance measurement The simultaneous measurement of absorbance and CD is complicated by the mechanism by which CD signals are optimized on Chirascan series spe
41. password of the Chirascan PC themselves APL Service Account The user account APLService has been created and given Administrative privileges by APL This account will be used by APL engineers during servicing of your spectrometer Do not remove this user or change the password Chirascan User Login and the Chirascan Users Group The user account Chirascan User has also been created by APL Chirascan series spectrometer users may login as Chirascan User or the system administrator may create individual user accounts A Chirascan Users group has also been created and users should be made members of this group If the Chirascan PC is connected to a network then this may affect the way in which users logon to the Chirascan PC The system administrator should ensure that network or domain users who want to use the Chirascan software are members of the Chirascan Users group Virus protection The HP Compag PC may be supplied with a trial version of a 3rd party anti virus package APL recommends that the Chirascan PC has up to date anti virus software installed at all times Most customers will have a preferred anti virus software package and APL generally recommends that the anti virus software which is supported by the customers ICT department be installed on the Chirascan PC However please see the section on installing 3rd Party software below Networking The Chirascan PC is supplied with networkin
42. sound and free from the distortion risks which accompany the use of other online filtering methods 6 5 4 Cosmetic smoothing The acquired spectrum will probably still exhibit a degree of wavelength dependent noise which can be further reduced for enhanced visualization and publication purposes using off line digital smoothing techniques The digital smoothing method available in the spectrometer software is based on the Savitzky Golay algorithm 4 Because such filtering methods can over smooth the spectrum distorting the underlying spectral features the smoothed results are always accompanied by a display of the residual spectrum which is the difference between the raw and smoothed data Any systematic trend in this residual plot is evidence of over smoothing and the result should be discarded A random residual indicates a smooth free from distortion Figure 6 7 smooth raw CD residual CD mdeg 160 190 200 210 220 230 240 250 260 Wavelength nm Figure 6 7 raw smoothed and residual CD data 6 6 References 1 PEM 90 Photoelastic Modulators Hinds Instrument Inc 3175 N W Aloclek Dr Hillsboro OR 97124 7135 USA 2 Drake A F Circular Dichroism in Methods in Molecular Biology 22 1994 219 3 Velluz L Legrand M Grosjean M Optical Circular Dichroism Principles Measurements and Applications Academic Press New York 1965 4 Press W Teukolsky S Vetterling W Flannery B Numerical Recipes in
43. subtract a single background from a number of spectra that are to be averaged Under these circumstances smoothing prevents reinforcement of the background noise in the final result Save and Print have their usual functions Click on OK to confirm your choice and close the dialog box or Cancel to close without implementing the new background Calibration is used to recalibrate the CD scaling factor and requires administrator privileges to gain access to it It should not be necessary to use it routinely and will be greyed out for the normal user Please refer to Section 4 15 1 if you wish to recalibrate the CD Document 4207Q108C03 01 Page 61 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences ee Deen RO F Chirascan Series User Manual 4 2 3 The Signal panel for Absorbance Signal Absorbance z 0 000774 AU hee Options Absorbance J Transmission Fluorescence PML Baseline Auto HY Background Recall Figure 4 6 the Signal panel with Absorbance selected When Absorbance is selected on the Signal panel Figure 4 6 the absorbance will be measured directly rather than derived from the HV signal if the Absorbance check box is ticked with circular dichroism selected The directly measured absorbance gives accurate values comparable to those obtained on a dedicated absorbance spectrometer but when this option selected the CD signal cannot be measured simultaneously The Options subpanel enab
44. to study chiral molecules of all types and sizes but it is in the study of large biological molecules where it finds its most important applications A primary use is in analysing the secondary structure or conformation of macromolecules particularly proteins and because secondary structure is sensitive to its environment e g temperature or pH circular dichroism can be used to observe how secondary structure changes with environmental conditions or on interaction with other molecules Structural kinetic and thermodynamic information about macromolecules can be derived from circular dichroism spectroscopy Measurements carried out in the visible and ultra violet region of the electro magnetic spectrum monitor electronic transitions and if the molecule under study contains chiral chromophores then one CPL state will be absorbed to a greater extent than the other and the CD signal over the corresponding wavelengths will be non zero A circular dichroism signal can be positive or negative depending on whether L CPL is absorbed to a greater extent than R CPL CD signal positive or to a lesser extent CD signal negative Circular dichroism spectra are measured using a circular dichroism spectrometer such as the Chirascan which is a highly specialised derivative of an ordinary absorption spectrometer CD spectrometers measure alternately the absorption of L and R CPL usually at a frequency of 50 kHz and then calculate the circular dichroism sign
45. wavelength nm is shown in Figure 2 30 The operating conditions are given in Table 2 11 C Program Files x86 Applied Photophysics Chirascan Examples R 3 Methylcyclopentanone dsa Circular Dichroism A File View Trace Math Scale Window Help aSamanw angang Circular Dichroism mdeg 220 240 260 Figure 2 30 CD spectrum of R 3 methylcyclopentanone Solvent Table 2 11 operating conditions for the spectrum in Figure 2 30 Document 4207Q108C03 01 BE 280 VVavelength nm Page 47 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences Sec AE P eas en Chirascan Series User Manual 2 10 Troubleshooting If the instrument does not operate properly please use the following table to eliminate any trivial causes of incorrect function There is also a frequently asked questions section on the APL website http www photophysics com faqch php to which you should refer if the table below does not help If the symptoms persist please contact Applied Photophysics Technical Support contact details at front of manual with a detailed description of your difficulty and the name of your spectrometer Each spectrometer has a name that can be found on the front of the monochromator Corrective action Instrument computer No power available Check that all power cables are cannot be powered on correctly plugged in and that the power at the
46. 00 iterations of Document 4207Q108C03 01 Page 95 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PPS re eh eS ae Chirascan Series User Manual the fitting algorithm may take some time In some instances the fitting does not converge because of the absence of a unique solution This too is symptomatic of an under determined model When you are satisfied that the fit is a good one and the calculated parameters are sensible you can print out a report by clicking on the printer icon The report displays the data the calculated fit data with residuals and the associated fitting parameters To plot the fitted curve in the Pro Data Viewer display click OK If you do not wish the residuals to be included on the plot clear the Include Residuals in Final Plot check box 5 3 3 Converting CD Units Select the traces whose units you wish to convert and open the Convert CD units dialog box using Convert on the Math menu Section 5 8 4 i on the graphical display toolbar Section 5 6 2 or the Convert button on the Trace Manipulation dialog box Section 4 3 1 Convert CD Units Convert From mdeg Convert To delta Figure 5 15 the Convert CD units dialog box The CD units of the selected trace are shown in the Convert From box the units you wish to Convert To are chosen from the drop down menu When converting between millidegrees mdeg and delta A a scaling factor is used and the conversion requires no further i
47. 1 to call up the Save As dialog box or Save selected traces on the File menu The Original raw data are never over written The results of any manipulation are stored in a new Datastore and a complete history of the manipulated traces is contained in the History of that Datastore which is opened on the View menu Section 5 7 2 5 4 Exporting data Right click on a filename in Pro Data viewer launchpad to display the File manipulation pop up menu shown in Figure 2 19 3 C Program Files Applied Photophysics EhirascansE sampl C Corrected lysozyme dex O m ge is Ci Lyso O 5m Open C Lyso 0 5m Browse with Explorer C Lyso Lem Export Data Decompose Data Delete Rename Properties Figure 5 17 File manipulation pop up menu Document 4207Q108C03 01 Page 97 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences Sect E AE A ER CAA Chirascan Series User Manual Select Export data to bring up the dropdown list in Figure 2 20 and choose an appropriate format for export to another program CSV format is suitable for third party programs such as Excel other options relate to formats derived from other APL products Select Data Format ProBinary ProD ataS tore APL ASCII JOSMP D 5 0 Simple FroD atal S Figure 5 18 options for exporting data 5 5 Handling multi dimension Datastores 5 5 1 Loading multi dimension Datastores In the example that follows an experiment was set up to me
48. 230 240 250 260 VVavelength nm Figure 5 4 CD spectrum of lysozyme with background selected The method of selection will depend on personal preference and on the number of spectra displayed For example if you wish to select one spectrum from several overlapping spectra using the dialog is easier than using the mouse pointer Once a spectrum is selected the Up Down arrow keys can be used to move the selection sequentially through the displayed spectra 5 2 3 Selecting more than one spectrum Selecting more than one spectrum or trace is often required for example to average several spectra Variations in the methods described in the Section 5 2 2 to select a single spectrum are used to select more than one e When using the mouse pointer to select spectra hold down the Shift key and click on the spectra of interest Clicking on the same spectrum twice with the Shift key depressed will first select it and then deselect it e When selecting from the drop down list hold down the Shift key and click on the filenames of interest Clicking on the same filename twice with the Shift key depressed will first select it and then deselect it e When selecting from the Trace manipulation dialog box Section 5 3 1 use the Ctrl and Shift keys in combination with the mouse to select filenames in the usual way Selected files are highlighted In general using the dialog box is the preferred method when dealing with more than one spectrum 5 2
49. 4 16 1 Fnidm Coil c w ral LU w oO 220 230 Wavelength nm Figure 5 48 calculated components of the secondary structure of lysozyme Document 4207Q108C03 01 Page 114 of 133 Applied hotop Ultrasensitive Spectroscopy for the Life Sciences Se EE Oa Chirascan Series User Manual The example given here is that of lysozyme Sample Files gt Lysozym txt The units delta epsilon are per amino acid residue Once you are familiar with the use of the program and what the results mean it is recommended that you read the notes that come with the program itself move on to your own samples 5 10 2 Using CDNN with your own results Measure the CD spectrum of your protein and carry out any manipulations such as averaging background correction and smoothing background correction is essential to get reasonable results Here we use the example of Lysozyme Circular Dichroism mdeg 180 190 200 210 220 230 240 250 260 Wavelength nm Figure 5 49 Lysozyme and background spectra several repeat measurements 0 2 mg ml Pathlength Molecular weight 14600 daltons Amino acid residues 129 Wavelength range 180 260 nm Bandwidth 1 nm Table 5 1 operating conditions for the spectra shown in Figure 5 49 Document 4207Q108C03 01 Page 115 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences Circular Dichroism mdeg 180 190 200 210 220 230 Wavelength nm 240 250
50. 6 2 Removal and replacement nssrrnisriiirsncessiernr Ennn En e a NEES EEEE ENNEN EEE 55 3 Maintenant nossen a ctw ae lige ane neta nec ita ENEA AAEE E EEan Aa hg EEan en a EEan a rin rE a 57 3 8 Moving INE SDECIIOMGICN iwiscsisaisiecierssessarstessatnwareei aii aana E a NATE Eei NE na EE ei 57 3 8 1 Parking the MION OC MON Wa LOl srrsarairesiss raan nE en E E TEET E E ATER E R 57 3 8 2 Lifting and moving the spectrometer sssssessssssersssererssserersssrrresssreressereresssrrressreresssreressereessseeeeesserres 57 4 PRO DATA CHIRASCAN ctiptnncnoannad ediemnashraeanosennanunct webiupiataedaast dia mentee oct nbndubteniaduentadianneatissanciaweiane nb eoweairenmenie ts 58 4 1 The Spectrometer COmirolP aie ha stctc ac jacececn tend shes sacen ceazdadetadenosdece pieddbentaaes see iedshues seein ladda oaeenin biedsbes a 58 42 The oignal DAMEN siess aa dete nbentte dua ae aaa raaa raa i 59 ANERE E N e EEE AE E T AE ce vance eas A aces and se ENEE E E EN E T 59 4 2 2 The Signal panel for Circular dichroism veeiscscevssnssdccnssassaveavsdvcdeossasesovseduvedoonssvusanssevaedence sancadsensamesseaesatsateas 60 A2 3 The Signal panel Tor AOS OD AICO lt apcicansstsnartantoxsassancastaneaceadanndoosiientonbeaneatontonstawsannadGansaeeeesenemeesasniiancessaneateed 62 4 24 The signal paneltor Vola E esaapee serene nctineaenareennan se eareencacese save ane woes ne pagan pe TEET 63 4 3 The Shutter attenuator panel ccccccssesececcccessec
51. A full description is given in Section 4 14 Launches Pro Data Viewer Chapter 5 P Calls up About Chirascan window which gives the Chirascan software version Click this icon and then on item of interest to call up the context sensitive help for that item Document 4207Q108C03 01 Page 71 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Pee Oe eee re Chirascan Series User Manual 4 12 Drop down menus Much of what is available in the drop down menus is more readily accessible from the icons in the toolbar A full list of the functionality is given below inevitably there is some repetition of the information in Section 4 11 4 12 1 The File menu Pro Data Chirascan EMULATION MODE View Configuration Help Open Ctrl O E i Open Examples Save Ctrl 5 Save As r Save Template As Exit 7 Te E r Figure 4 21 the File menu The File menu Figure 4 21 presents the usual options and shortcuts for opening and closing files It is unlikely that you will need to use these because their functionality is more conveniently accessible elsewhere in Pro Data and in Pro Data viewer The examples folder can be accessed from this menu in which there are several examples of spectra supplied by APL that can be used as templates to set up Pro Data or as tools to familiarise oneself with the use of Pro Data viewer 4 12 2 The View menu Pro Data Chirascan EMULA
52. AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences User Manual Chirascan Series Spectrometers Chirascan and Chirascan plus Circular Dichroism Spectrometers February 2012 Document 4207Q108C03 01 Applied Photophysics Ltd 21 Mole Business Park Leatherhead Surrey KT22 7BA UK Tel UK 44 1372 386 537 Tel USA 1 800 543 4130 Fax 44 1372 386 477 Applied Photophysics was established in 1971 by The Royal Institution of Great Britain WWW ohoto ohysics Corn Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences eis ciate orate fe Pret ERR E ESR Chirascan Series User Manual This page is intentionally left blank Document 4207Q108C03 01 Page 2 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PE ne oR aan ro Chirascan Series User Manual This document contains important safety information Read this document before attempting to install or use the Chirascan or Chirascan plus spectrometer Failure to do so could result in death or serious injury Document 4207Q108C03 01 Page 3 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PEE a eee eee Chirascan Series User Manual USE OF THIS DOCUMENT This document is intended to inform the operator of Applied Photophysics Chirascan series Chirascan and Chirascan plus spectrometers on their design installation and operation Some of the items that may be supplied with the spectrometer
53. CD data 200 0 ccccecccccccceeceeeeeeeeeeeeeeeaeeeeeeeeeeeeeaaesceeeeeeeeeeaeaaeeeeseeeesaaaaaeees 133 Document 4207Q108C03 01 Page 18 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PASAMAN SRDRTaRRUESEL INTRA Chirascan Series User Manual TABLES PNE TENDU I TOW aS r paacca cece acecsoncteauc ot aeneaeeieeso E T E E S 23 Table 2 2 maximum bandwidth at low wavelengths cccccccccceeecceceeeeeeceeeeeeeceeeeeeceeeeeesseueeesseaeeeessaeeessaneeeesaaeees 38 Table 2 3 recommended conditions for measuring protein CD spectra ccceeccccceeecceceeeeceeceeseeeceeeeeeseeeeeessaneees 39 Table 2 4 operating conditions for the spectrum in Figure 2 23 nnsnannnunnnnnnnnennnnnnnnnnnornrnnnrrnnrrrnnrrnnrrnnrrrnrerenenne 40 Table 2 5 operating conditions for the spectrum in Figure 2 24 oannsnannnennnnnnnnnnnnnnnnnnnnornrnnnrrsnrronnrnnnrrnnrrrnrerenenne 41 Table 2 6 operating conditions for the spectrum in Figure 2 25 nsnannnunnnnnnnnenrnnnnnnnnnnnrrnnnrnnnrrrnnrrnnrrnnrrrnnernnenne 42 Table 2 7 operating conditions for the spectrum in Figure 2 26 nsnannnennnnnnnnnnnnnnnnnnnnonnrnnnrnsnrrrnnrrnnrrnnrrrnnernnenne 43 Table 2 8 operating conditions for the spectrum in Figure 2 27 s nnsnnnnnunnnnnnnnunnnnnnnnnnnnrnrnnnrnnnrronnrnnnrrnnrrrnrrrnnenne 44 Table 2 9 operating conditions for the spectrum in Figure 2 28 nnnnannnunnnnnnnnunnnnnnnnnnnnrnrnnnrnnnrrrnnrrnnrrnnrrr
54. D O Celf teszt Passed Advanced Figure 4 29 the CD DC photometric Channel window The Chirascan and Chirascan plus are CD spectrometers and therefore this channel and the CD AC channel will always be active Chirascan On the Chirascan The CD DC Photometric Channel dialog permits you to apply voltages to the CD detector which govern its gain The PMT HV slider allows you to choose an arbitrary detector HV between 0 and 1000 volts the ZeroHV button will set the voltage to zero the AutoPM button will adjust the detector HV so that the photomultiplier anode current resulting from the intensity seen by the detector is at the optimum level for the Chirascan electronics Click on the Apply button to apply the HV to the detector In a CD scan the detector HV and so its gain is adjusted so that at every point in the spectrum the anode current is at its optimum level for the Chirascan electronics If this window were open during a CD scan the PMT HV slider and the associated edit box would show HVs that changed as the scan progressed being high where the sample absorbance Is high and low where the absorbance is low Chirascan plus On the Chirascan plus the way the solid state LAAPD CD detector works is quite different from the photomultiplier tuibe PMT detector use don the standard Chirascan A PMT has a variable voltage applied to the anodes which varies the gain of the detector optimizing the recorded CDDC and CDAC signals T
55. INED IN THE LICENCE AGREEMENT THESE INCLUDE THAT THE LICENSEE MAY NOT SELL RENT LOAN OR OTHERWISE ENCUMBER OR TRANSFER LICENSED SOFTWARE IN WHOLE OR IN PART TO ANY THIRD PARTY FOR A FULL COPY OF THE LICENCE PLEASE CONTACT APL OR SEE THE SOFTWARE INSTALLATION DISC Chirascan and Chirascan plus are trademarks of Applied Photophysics Ltd Microsoft Windows and Excel are registered trademarks of Microsoft Corporation in the United States and other countries Adobe and Reader are registered trademarks of Adobe Systems Incorporated in the United States and or other countries Norton is a trademark of Symantec Corporation or its affiliates in the United States and other countries McAfee is a registered trademark of McAfee Inc in the United States and other countries Sophos is a registered trademark of Sophos plc and Sophos Group Cary and Agilent are registered trademarks of Agilent Technologies Inc Julabo is a registered trademark of Julabo Labortechnik GmbH Sigma Aldrich is a registered trademark of Sigma Aldrich Co Missouri United States All other trademarks or registered trademarks are the sole property of their respective owners Document 4207Q108C03 01 Page 4 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eee ops een 32 Chirascan Series User Manual HAZARD AND OTHER INDICATORS HAZARD INDICATORS USED IN THIS DOCUMENT A CAUTION The sign to the left is used to indicate
56. Lyso 0_5mm PLO004 dsa Circular D C 6 E File View Trace Math Scale Window Help 7 Circular Dichroism mdeg 180 180 200 dg s aaro aag a a a m CS G 2D Co 210 220 230 240 250 260 VVavelength nm Ready Figure 2 17 smoothed and background corrected spectrum of lysozyme red and smoothing residuals cyan Note that even when the Trace Manipulation dialog box is closed any removed traces are remembered Should you wish to carry out further manipulation simply re open the dialog box and all the traces will be listed whether visible or removed enabling you to carry on where you left off Document 4207Q108C03 01 Page 34 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences PE E AE AAO ete Chirascan Series User Manual 2 6 Saving exporting and converting data 2 6 1 Saving data If you have performed operations in Pro Data Viewer e g smoothing averaging or background subtraction added comments or altered the data in any other way you may want to save the results of the manipulation The manipulated data are not saved automatically To save the result go to File gt Save Current Plot keyboard shortcut Shift S to call up the Save As dialog box or File gt Save selected traces Ctrl Shift S The original raw data are never over written The results of any manipulation are stored in a new Datastore anda complete history of th
57. Panel SCP Local D Remote Figure 5 31 the Connect dialog box The client server architecture permits remote monitoring of experiments in progress If a copy of Pro Data Viewer is installed on a remote computer establishing a client server link established using the Remote setting will enable you to follow an experiment on the second computer The IP address of the Chirascan computer will not be the same as the default local IP address your network administrator will be able to inform you If the address is correct and the instrument is running the Pro Data Viewer display panel will open Pro Data Viewer will listen to Chirascan and report its status behaving as a real time display If the address in incorrect the error message shown in Figure 5 32 will appear Chirascan gt Sorry The Chirascan Viewer application lost its link to the Control Panel at IP Address 127 0 0 1 Please make sure Chirascan Control Panel is up and running and the IP Address is correct and then try to connect again Try to reconnect Figure 5 32 the Connection error message e Allow Cary files allows files imported from some Agilent Cary series spectrometers to be plotted using Pro Data Viewer 5 7 5 The Help menu The Help menu Figure 5 33 gives access to the Pro Data Viewer launchpad help system Ta Pro Data Viewer File View Directory Preferences Windows Help Topics C Program Files K86 Lys
58. Scale Window Help x G68 8rR OH O OOHOO H O 0 8 8 h c 160 140 120 100 80 60 40 20 Circular Dichroism mdeg 20 40 250 300 350 400 450 500 550 600 650 700 750 a00 VVavelength nm Figure 2 28 CD spectrum of tris ethylene diamine cobalt chloride Table 2 9 operating conditions for the spectrum in Figure 2 28 Document 4207Q108C03 01 Page 45 of 133 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences EE E AE ET A 5 aos Chirascan Series User Manual 2 9 7 Camphor sulphonic acid The CD spectrum of 1 S camphor 10 sulphonic acid plotted as CD mdeg against wavelength nm is shown in Figure 2 9 The operating conditions are given in Table 2 10 F C Program Files x86 Applied Photophysics Chirascan Examples Camphor sulphonic acid dsa Circular Dichroism A File View Trace Math Scale Window Help al x GS i 2 m S 9 Circular Dichroism mdeg 180 200 220 240 260 260 300 VVavelength nm Ready NUM O E Figure 2 29 CD spectrum of camphor sulphonic acid Time per point Table 2 10 operating conditions for the spectrum in Figure 2 29 Document 4207Q108C03 01 Page 46 of 133 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences 2 9 8 R 3 methylecyclopentanone Chirascan Series User Manual The CD spectrum of R 3 methylcyclopentanone plotted as CD mdeg against
59. TION MODE File Configuration Help Pro Data Viewer Devices Remarks Toolbar Always on Top Figure 4 22 the View menu The View menu Figure 4 22 offers five options e ProData Viewer launches ProData Viewer Chapter 5 e Devices opens the Devices Window This contains a number of icons corresponding to hardware devices that may need to have certain parameters adjusted See Section 4 13 for more details e Remarks opens the Remarks and Comments window in which you can record information about your experiments The remarks are stored in the Datastore and can be viewed in Pro Data viewer e Toolbar a toggle to show or hide the toolbar e Always on top a toggle to ensure that Pro Data Chirascan is always visible Document 4207Q108C03 01 Page 72 of 133 AppliedP hotophysics Ultrasensitive Spectroscopy for the Life Sciences eee eee eee eee eee eee eee ee eee ee Chirascan Series User Manual Change Sample Handling Unit SHU Access Control Signal Preferences Ctrl k Figure 4 23 the Configuration menu The Configuration menu Figure 4 23 offers three options e Change Sample Handling Unit SHU opens the Select SHU dialog box Figure 4 24 Unless the Kinetics Sample Handling Unit CS SF is fitted Equilibrium Sample Handling Unit should be selected Select SHU Equilibrium Sample Handling Unit ESHU O Kinetics Sample Han
60. Triggered Spectrum 0 Kinetics l Acquire Pause 0 Spectra Kinetics Timed intervals 0 1 m Progress and Status Ready For Help press F1 Figure 2 4 the Pro Data SCP The Pro Data interface enables you to configure the instrument and to manage experiments It is divided into a number of panels Signal Temperature Control Unit Sample Handling Unit Monochromator Sampling Sequencer and Progress and Status in which are grouped the parameters relevant to the control or management of that panel s function In addition there is an unlabelled panel for controlling shutter function and detector high voltage Document 4207Q108C03 01 Page 25 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences AE S Oe 3 Chirascan Series User Manual 2 4 Measuring a CD background and spectrum When a sample is placed into the sample chamber it is inevitable that a small quantity of air will be introduced to the low volume light path within the chamber It takes only a few seconds to re establish an air free environment and you should not need to wait longer than that before starting your measurement 2 4 1 The CD background Every CD spectrometer has a CD background caused by the interaction of circularly polarized light CPL with the slight birefringence found in the PMT detector window and to a lesser extent in the cuvette To see the true CD spectrum of your sample this background must be measured and subtracted
61. a YMawelengtt Cell Cell Wavelength Temperature Figure 5 20 the dimension menu In this case wavelength in the X column and Temperature in the Z column are highlighted indicating that in the display above wavelength is the x axis and the CD spectra at different temperatures are projected down the z axis onto the display The third dimension is shown in the slider bar To change the display select a different combination of x and z axes from the menu for example Wavelength and Cell Temperature will appear as the third dimension in the slider bar Figure 5 21 r U My Data Turret testing Turret 4 CSA 4 cells0043 dsx Circular Dichroism A File View Trace Math Scale Window Help x g amp n Gee CoD CE Temperature A D v 0 E z a Q i T g Q 3 U O 260 270 280 290 300 310 320 330 VVavelength nm Figure 5 21 CD spectrum for each of the four cells at 20 C Spectra for the four cells can be displayed at any one of the temperatures at which they were measured simply by selecting the temperature from the dimension bar above the display Once data are displayed they can be manipulated as previously described in this Chapter Document 4207Q108C03 01 Page 99 of 133 AppliedPhotophysi Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual 5 5 3 Mining multi dimension Datastores Multi dimension Datastores can be mined
62. a generating formats that are suitable for third party applications Document 4207Q108C03 01 Page 86 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences E TIAE E Chirascan Series User Manual 5 1 3 Viewing experimental conditions Experimental parameters wavelength range step size time per point bandwidth plus sample information provided by the user via the Setup dialog box in the spectrometer control panel sample description concentration cell type and pathlength are stored with the results file and can be viewed on the Datastores Details View the area to the bottom of the launchpad Figure 5 2 The details shown will be those of the file highlighted in the launchpad file list u File Chirascan test data dsx ProBinary ttributes Time Stamp Thu Feb 25 12 26 06 2010 File ID EGDF6CA6 B630 4cba A1CA B6 f80D66F10 Is CFR Compliant false Original unaltered data Remarks Ready Off line Figure 5 2 the Datastores Details View 5 2 Displaying and selecting data 5 2 1 The graphical display One or more files can be displayed in the graphical display e To display a single file double click on the filename or choose Open from the File menu Section 5 7 1 e To add a file to the graphical display drag it onto the display or right click and choose Add DataStore to Graph e To add more than one file at a time select multiple files from the list in the usual way using the mouse po
63. a a TA AREE Ea 22 22 Fe OW CNG Oei E A E E E S 23 2 3 Launching the SoftWare ccccccsesscecceeseeeceeceeceeseecseueeecsaececsageeeeseuseeessageeecsaeeeeseaseesseageeessageeessageeessaags 25 2 4 Measuring a CD background and spectrum cccccccccseeseececeeeseceecceeeececceseuseeeeeseeaeeeeeseuaaeeeeesseageeeessaaaaes 26 2A A CED PAIE O e E E E E E E E tas 26 LALE E LMM e A E E A A E E R 28 2 5 Viewing and manipulating spectra sisarcevirssesicteeucewveieecivdeduceandviveniiveaverewivinvedy iisuesaudennbaeadids puvaveddaneividaisanedeans 29 DUM VOW E Tea a EEE E EEE E ET ET 30 TaZ Compare SPEC er EE E A EE eeaane een ee 31 2 5 3 Manip lating SPECE aA aaririsecuriserinikaredrinied enkarna AEE aaar ener aetancnsauavatsseessarnancuaeacouearenteseateeaetan 31 2 6 Saving exporting and converting DAtA cccccccccssssccceceeeeeceeeceeeeeceeccseaeceeesueaseeeesseaueeeesssaaseeeesseaaeeeesesaaaes 35 PESEE T A E e A E A E E A VA E E E E E E E E 35 2 6 2 Exporting data from Pro Data ViQWEL ssscccccssseccccseseccccseescceceeesseceessausecesseeaecesseeaecessuaaeeeessaaaeeeeees 35 2 6 3 Converting data for use with third party Programs ccccccccsssscccccessecccceesscceccsueececsaeasecessaaaeeeeesaaeeeeees 36 2d FANU e E E 37 2 8 Operating Noles and NNS esisscesasoniiviriinyn niei i ai a i ani a iei aiai 37 PO SOIC CIN SUC 51 ZO 59 pusoanantandessaguane tone dgeausanudonnuoeaisdaipeamescuntssents ana seosanba
64. aeeeeseaeeeesseeesens 90 Figure 5 7 example menu of properties that can be plotted AS Y AXIS cee eeccecceeeeceeeeeeeeeeeeeeeaeeeeeeeesaaeeeeseesaaeeees 90 Figure 5 8 absorbance spectra for Water red cccccceeseccecceeeeceeeeueeeeeeeeseaeeeeeeesseeeeeesseaaeceeeesaaeeceeessaaeeeeesssaeeeees 91 Figure 5 9 the Trace Manipulation dialog DOX ccccseeccccsseeeeeeeeeeeceeeeeeeeeeeeeeseeeeeeeaueeesseeeeesseeeeessaeeeeeseaeeeesseeeenens 92 Figure 5 10 raw data with dialog box Called UD vec csscdonaceaccsiais oncdicsassetnednelaceqsesachecuasinunaesueieacdodiaeucactencnelemsuarwen 92 Figure 5 11 An appropriate smooth left and an inappropriate one right cceecccecseeeeecseeeeeeeeeeeeeeeseeesneeeeeens 93 Figure 5 12 Trace manipulation dialog box after spectrum manipulation eee c cece ceeeeeeeeeeeeeeeeeeeeeeaeeeeeeenaaeeees 94 Figure 5 13 smoothed and background corrected spectrum of lysozyme red and smoothing residuals blue 94 Figure 5 14 hydrolysis of glucuronolactone with potassium hydroxide ccccceeeeceeeceeeeeeeeeeeeeeeeeeeeaaaeeeeeeesaneeeess 95 Figure 5 15 the Convert CD units dialog DOX ce eecccceececeecceeeseeeeeeaeeeeeeeesaeeseeeeeesaeeeeeesseaseeeseeseeeeceeeeseaeseeessaeaeeeees 96 Figure 5 16 Converting to a normalized UN It cece cccccseeeeceeecaeeeeceeceaeeeeeeeecaeeeeeeeseeaseeeesseeseeeeeesaeeeeeessaaeeeeeessaeeeees 97 Figure 5 17 File Manipulation POP UP MENU
65. age 36 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences Re eae ERP Chirascan Series User Manual Save in ProBinarys format Save in ProBinary format Acom ProD ata binary format Save in ProDataStore format Acom Proll ata ASCII format Save in ProDatals spread sheet friendly CS format E Save in Pro k 7 Pro k Il format AFL ASCII SAPL ASCI SPECTREINE TIC Save az ab Separated text file r Save m JCAMP D format Use sensible file extensions e g CS data sawed with CSY extension Remove Acorn file type information from filename e g Lys B FFA gt LysB ce Delete original DataStore file Silently overwrite existing files Skip unrecognised or unreadable files silently Cancel Figure 2 22 the APLDataConverter Option dialog box 2 Printing The Print icon in Pro Data viewer will print the current Pro Data viewer display 2 8 Operating notes and hints 2 8 1 Selecting Step Size SS The step size will determine the number of data points that make up a spectrum For a given time per point larger step sizes will reduce the overall scan time of an experiment but will also reduce the resolution of the spectrum smaller step sizes will improve the resolution of a spectrum but will increase the experimental time An appropriate step size can be chosen using SS FWHH 20 where FWHH refers to the full width at half height in nm of the narrowest peak in the spectrum If this is
66. al Document 4207Q108C03 01 Page 20 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual From its launch in 2004 the Chirascan led the world in setting the standard for steady state circular dichroism spectroscopy That lead was extended by the launch of the Chirascan plus in 2006 which uses a large area photodiode LAAPD based detector with higher quantum efficiency than the photomultiplier tube PMT detector used on the standard Chirascan Developed with innovative optical design features to maximise light throughput particularly in the far ultraviolet UV wavelength region and a digital data acquisition system that facilitates collection of accurate and precise spectra the Chirascan and its sister instrument the Chirascan plus are able to produce data unrivalled by any other commercially available circular dichroism spectrometer Document 4207Q108C03 01 Page 21 of 133 Applied hotophysi Ultrasensitive Spectroscopy for the Life Sciences Spc AE A OER 5 ep Chirascan Series User Manual 2 QUICK START This Chapter describes the basic operation of the spectrometer covering the start up of hardware and software use of the spectrometer instrument control and data acquisition software to collect CD spectra and backgrounds use of the graphics software to carry out common operations on the data and to save data appropriately and conversion of data formats for export to third party programs Mo
67. all powered on Document 4207Q108C03 01 Page 23 of 133 AppliedP hotophysics Ultrasensitive Spectroscopy for the Life Sciences Fe eS Ste eee Oe ae eae Chirascan Series User Manual Figure 2 2 the system panel Once the lamp is struck it is strongly recommended that it be left on for the duration of the work planned for the day Repeated powering on and powering off will lead to instabilities see previous paragraph and will reduce the life of the lamp However if it is necessary to restart the lamp after running it must be allowed to cool for about 15 minutes before re ignition is attempted The light emitting diodes LED s on the System panel Figure 2 2 are labelled Status Tx and Rx Tx and Rx will illuminate when commands are being transmitted or data is being received respectively the green Status LED should be constantly illuminated If it is flashing it indicates that one or more of the electronics self tests has failed If this happens try restarting the electronics and if the problem persists please contact APL technical support Figure 2 3 the lamp panel The red LED on the lamp panel Figure 2 3 indicates that power is present The digital timer records the number of hours that the lamp has been running adjacent to the timer is a recessed reset button the timer should be reset when the lamp is changed Document 4207Q108C03 01 Page 24 of 133 Applied Ultrasensitive Spectroscopy for the Life Scienc
68. anaii onnaa seiis 124 6 3 Simultaneous calculation of absorbance and CD cccceeecccccsseeeeeeeceaeeeeeeesaaeeeeeeeseeaeeeeeessaaeeesesseaeeeeeeeas 125 SA WE eT E LION PA IEI EE E E A E E A S AEE T 125 6 3 2 Absorbance measurement nE a aE ae vo EEE E NENE AEAEE E NEERA 126 6 4 Adaptive AON Seca ndisare te teeter nde tuceonajanasseddi fe slence eneisense stint csc acne catenin aaleataneead ghar te tnieatent adieaitigesienCanandiemaneeed 128 oe i Miguigele leis pee eee eh ane ee ke ET E ee eee 128 GAZ TS FO PII MAIC EOD ea T E E E E E E A 129 6 4 3 The adaptive sampling algorithm ssssseesssssenesssrrrssserersssererssssrresssreresssrerossereressrereessreressereresserreessereees 129 EKSA Aled MIC HIG EEr A E A E E E E E A E E E E E O E ET 132 ks WIE OCUIC O eesin E EE E E E 132 6522 Electronic ILErNE sasiosteassarcaeeatoncdacareancsinassesiactiunssascntoasanebnceestuuenstaweasenatdaseatoaiasnienseaudacdaceevenenesdesaueanesevaieas 132 Co SENAVE ENE a E E T E O E E 132 CaA Cos menco gilele UNE srei riena a EE E E E rR E E EEN 133 G30 IB SIO V CNC SS nre EE AE E AEE 133 Document 4207Q108C03 01 Page 15 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences pRSAnes oReRtenersetenens Chirascan Series User Manual FIGURES Figure 1 1 the Chirascan plus spectrophotometer ccccccsessecccccseseeceecceeeeeeeecsuauseeeeeeeeuseeeeeseaeceeessaaseeeesesaaeeeees 20 Figure 2 1 the purge gas manifold gas flow
69. and activated charcoal filter the purge gas Is split three ways by a manifold to provide separate supplies to the lamp housing monochromator and ESHU The flow rate for each supply is set at the manifold as described in Section 2 1 lt is not normally necessary to disconnect the supply to the lamp housing and monochromator except when moving the spectrometer but if the ESHU is to be removed the supply to the ESHU first should be disconnected as follows 1 Ensure that there is no purge gas flow to the ESHU 2 Unscrew the compression fitting nut connecting the purge gas supply line to the rear of the ESHU Figure 3 3 3 Withdraw the stainless steel supply line and move it away from the ESHU taking care not to kink the line Purge gas inlet Figure 3 3 the purge gas inlet connection on the rear of the ESHU 3 4 Light source 3 4 1 General WARNING The spectrometer is equipped with a light source 150 watt xenon or mercury xenon arc lamp that produces intense ultraviolet radiation that can irritate the eyes and may impair eyesight Never look directly at the light source Do not open the lamp housing while the lamp is operating or immediately after it is powered off Do not allow the skin to be exposed to UV radiation The spectrometer uses an air cooled 150 watt xenon Xe or mercury xenon Hg Xe arc lamp as a light source The housing is designed for the lamp to be easily removed and replaced without the need for realignm
70. anel for Voltage Note that this panel is intended for diagnostic purposes only It is unlikely that you will need to use it in the normal course of events Signal Voltage a077 Channel Eie PMU AuoPM Aux 2 PML AutoPM Baru pmo Autor Waicopc pMu AutopM 1 5 CDAC Figure 4 7 the Signal panel with Voltage selected The five signal channels currently assigned are auxiliary1 auxiliary2 fluorescence circular dichroism DC and circular dichroism AC Only those that are actually installed will be highlighted in the Voltage panel Figure 4 7 the others will be greyed out When in voltage mode channels 1 to 4 operate in a similar fashion each provides a voltage scan proportional to the intensity of a signal falling on the detector photocathode at a fixed detector high voltage This allows sensitivity checks and elementary voltage scans to be performed for diagnostic purposes For example you could use the fluorescence channel to determine where the maximum intensity of a fluorescing sample occurs or the CD DC channel in the absence of a sample to look at the lamp profile Channel 5 the CD AC channel gives access to the voltage arising from the polarization modulation induced by the photo elastic modulator It is non zero only in the presence of a sample with a chiral chromophore or if a birefringent medium is placed in the path of the modulated CPL The information measured with this channel is subs
71. ase M1 M2 button Table 3 1 module labels for the standard configuration Some of the standard ports are used for internal connections or are unused others are for external connections The upper port on the USB module labelled DRIVE is currently unused and is intended for future development The lower port labelled TEMP INPUT is a temperature read port the connection cable splits and goes partly to an internal connection and partly to the ESHU If the ESHU is removed from the spectrometer it can be disconnected but there is no need to remove the cable from the electronics rack The bottom port is for the USB connection to the PC On the Chirascan the lower port on the PEM module is for a PMT detector This is normally a CD detector but may be a fluorescence or fluorescence polarisation detector On the Chirascan plus the port is for an LAAPD CD detector only The lower port on CONTROL module M2 labelled DRIVE 2 connects to the emission monochromator of the CS SEM accessory The remaining ports are for internal connections Document 4207Q108C03 01 Page 50 of 133 Ultrasensitive Spectroscopy for the Life Sciences PEREA a a Re Cr Chirascan Series User Manual 3 3 Nitrogen purge gas connections The reasons for purging the spectrometer with nitrogen when using an ozone producing lamp and settings for the purge gas flow rates are described in Section 2 1 of this User Manual After passing through an 13x molecular sieve
72. aseeesseeeeneas 66 POUC A le al OIG DANO N a EE E EEEE cua TNE A 66 Figure 4 13 the Sampling Advanced dialog DOX ccccccccsseseceeceeeeeeeeeeeeeeseeeesseeesceeeeseaeceeeesaeeeeeeessaaeeeeeesseaeeeees 67 Figure 4 14 the Disable oversampling warning message ccccccseseeeceeceeeeceeeeceeeceeessaeeseeeeesaeeeceeessaaeeeeeessaeeeees 67 Figure 4 15 th Sequencer panel ciccccarsncctvacaresnonednsadvannenasevnaessduaaedacnedede sade doncsasdiassenedy esi ockededacededcnesendebsessiicneeabenedd 68 Figure 4 16 the Progress and Status panel ccccccsssseccccceeseeceeecueeeeeeceeaueeeeeseeeeeeeeseeaaeeeeessaaeeeeesseaeeeeessaaeeeess 68 Figure 4 17 the Temperature Control Unit panel cccccccssececceeseeeceeeeeecseeececeeueeessegeeessaaeeeeeaseeesseseesseeeeeees 69 Figure 4 18 the Sample Handling Unit panel cccccccccccssseeeeeeeeeeeeeeeeeeeeeeeeeeseaesseeeeseaeseeeeeseaeeeeeessaaeeeesessaaeeeees 69 Figure 4 19 the Sample and Cell description dialog DOX eeeccecccseeeeceeeceeeeeeeeeeeesceeeseeeeeeeeeseeeeseeessaaeeeeeessaaeeeees 70 Figure 4 20 the SCP Toolbar cccccccccsssseecccceseceecceeesececcceauececcssauseeeesseauseeecsssaaeeeecsseaaeeeeessaageeeeessaaseeeesssagaeeees 71 PIQUE 2 1 the PISMENU susie cnesassanensgivernctqrnateadans EEE EEEE 72 Figure 4 22 the VIEW MCU iinccsnsensorndiiisdnaetdusqounetnedescbwrevaxhiedsduocesisneanetusduncheuvouesbbdsduarbieagemauasisuchautenenbada
73. asure a CD spectrum of 1 S camphor 10 sulfonic acid from 240 nm to 340 nm for each of four cells mounted in a CS PCM 4 cell auto changer at temperature intervals of 5 C from 20 C to 80 C The three independent variables make it a three dimension dataset Load the file in the usual way from the Pro Data Viewer launchpad to display a set of CD spectra one for each temperature for cell position 1 The Viewer display is subtly different when a Datastore of more than two dimensions is displayed a slider bar representing the third dimension in this case cell position is added beneath the toolbar Figure 5 19 The spectra from each cell can be displayed by moving the slider to the appropriate position on the bar f U My Data Turret testing Turret 4 CSA 4 cells0043 dsx Circular Dichroism eon x A File View Trace Math Scale Window Help ox EELE CoD CD Cell i 1 000000 35 30 0 v T E 25 z 20 Q Lc 15 Q 6 10 J U O 5 0 240 250 260 270 280 290 300 310 320 330 340 VVavelength nm Ready NUM Figure 5 19 Stepped temperature ramp showing first cell CD spectra Document 4207Q108C03 01 Page 98 of 133 AppliedPhotophysix Ultrasensitive Spectroscopy for the Life Sciences Spc E AE A ER AA Chirascan Series User Manual 5 5 2 Changing the display axes Right click on the x axis legend to display the dimension menu Figure 5 20 r 1 and lt Ass Options
74. cccceccccaescccceseccceueccseuuseesauecesseecessaueceseuecessaaecessaueeesaesessaages 89 5 2 4 Zooming and re scaling the display USING the MOUSEC ccccsssseccccceeececcceeeccesseeseccesseeueceesseeaeeeessaaeeeeees 89 5 2 5 Displaying different properties cceecccccsssseccccceesecccceeesecceceaeecceesseeceeeseaeceessaaaeceesseeaseeessageeeeessagaeeeeees 90 5 3 Manipulating Fitting and Converting data ccccccccccssseseceeceeeeeceeeeaeeseeeeeseeeeeeeeesseaeeeessaeaeeeeesssaeeeeesaaaess 91 yee Me TV ea NUE IE Ol Vis ccsrsests asco csrsecwrasisegane E A E E E 91 BS I IS E E sora S E E E A E E E E E 95 Be C OTTE C IES eoa A E E N E O 96 ao Pe e eig 6 2 1 P E E N T E E S E E ee eee eee 97 DA OUI AN eee E EE E EEE E E E E E EEE EE E 97 5 5 Handling multi dimension Datastores cccccccssececceeececcsseeccseuseecceuseeessuseeecsaueeeeseaseeeseseeessagseessaneeesseaes 98 5 5 1 Loading multi dimension DatastoreS ccccccsssscccsseccccenscccseueccssueeecsescecseecesseueceseuecesseuecessaueeessaesessanees 98 5 5 2 Changing the display AX SS nistiassssrssortinns aa aaaea Ea EAE 99 5 5 3 Mining mu lti dimension DAataStOLeS ssrsisrseripssissran anoa a n E E ANET EENEN N AES 100 S6 FODA VEW CODI CONG orinar an aO EEEE EE T En 101 5 6 1 Launchpad toolbar iCONS ccsssscccccssssccccceesecceccueseccesaeesececseueeeceeseeaeeceesauasecesseeaeeeeessensecessuaneeeessagaeseeees 101 5 6 2 Graphical display too
75. ch appears after Acquire has been clicked will abort the acquisition clicking Pause will stop the acquisition without aborting and Resume which appears after Pause has been clicked will restart the acquisition from the point at which it was paused 4 8 The Progress and Status panel Progress and Status AutoPM ing _ Figure 4 16 the Progress and Status panel The Progress and Status panel Figure 4 16 provides information concerning the current status of the spectrometer and of data acquisition The box on the left of the panel will give the status e g Ready Stirring Acquiring the two bars to the right of the panel give a visual representation of the progress Document 4207Q108C03 01 Page 68 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual 4 9 The Temperature Control Unit panel Temperature Control Unit QN Temperature C 19 99 T Ramp Disabled Figure 4 17 the Temperature Control Unit panel The Temperature Control Unit panel Figure 4 17 is disabled unless a temperature control device such as the CS PCS single cell controller or CS PCM 4 cell autochanger is fitted For further information see the User Manual for the fitted device 4 10 The Sample Handling Unit panel ESHU panel Sample Handling Unit ESHU Temperature C 2r B Titration Shirrer Figure 4 18 the Sample Handling Unit panel The Sample Handling U
76. controllers ccccceccceeeeeeeceeeeeeeeeeceeeeceeeeseeeeeeeeesseeeeeeeesaaeeeeeessaaeeeees 22 PIES 22 IS SYS MN ANG baa c AE E E A ET tada caine acer E E T T 24 Foue 2532 1S Ne panel sesia erni Eseia aa EAE Ea SEAE ORE aia i a ESEE ARRE 24 Figure 24 th Pro Data SOP acete ce secsn ceicsroscneeincdbssintensasinenstbeasdantiaddisnaddestintinstadsincesideainsdossadsosicstincitenencaddadasidhesutonexsccnecbastscs 25 Figure 2 5 the signal panel in the Pro Data interface ccccceeccecceeeeeeceeeeeeceeeeeeeeeseeeeeeeeesseaeeeesaeeesseseeesseeeeneas 26 Figure 2 6 Monochromator panel in the Pro Data interface ccccccccseeeeeceeeeeeeeeeseeeeseeeeesseeeeeesaeeeeseaeeeesaneeeens 26 Figure 2 7 Sampling panel in the Pro Data INterface cccccccccccssseeeeeeeeeeeeeeeecueeeceeesseeeeeeeeseaeeeeeessaaeeeeesssaaseeees 27 Figure 2 8 Background CD spectrum ccccccsssseccecceeesecececeeaseceeecsaseeeeeeuaaeeeeseeaaeeeeeseeaaeceeesseaeeeeessaaseeeeessaeeeees 28 Figure 2 9 the File Names dialog DOX cccccccsssseeeecceeeecececeeesecececauseeeeesseaaseeeesssaasceeesseaaeeeeessaaaeceesssaaeeeeesssageeeees 28 Figure 2 10 CD spectrum of lysozyme raw data ccccccccccseeeeceeeeseeeeeeeeeeaeeeeeeesseeseeeesseeeeeeeessaeeeeeesseaeeeeeessaeeeees 29 Figure 2 11 Pro Data Viewer showing the launchpad left and graphical display right ceeseeeeeeeeeeeeees 30 Figure 2 12 CD spectrum of lysozyme
77. cope style live signal display that can be launched by clicking on the icon in the top right corner of the panel Figure 4 3 The units will be those of the chosen measurement mode an instantaneous value of the measurement is also displayed in the box to the right of the drop down menu Live Signal Figure 4 3 the Live Signal display Document 4207Q108C03 01 Page 59 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PS Re eee Chirascan Series User Manual 4 2 2 The Signal panel for Circular dichroism Signal Circular Dichroism 415 1291 mdeg Options EE 5 delta A d Absorbance milidegrees C Fluorescence AutoPM Baseline Auto Subtraction Show Calibration Figure 4 4 the Signal panel with Circular Dichroism selected The most commonly used mode of data collection is circular dichroism When this option is selected the Signal panel has two sub panels Options and Background Figure 4 4 On the Options subpanel choose either delta A delta absorbance or millidegrees for the units of the CD measurement The AutoPM button will adjust the detector HV so that the photomultiplier anode current resulting from the intensity seen by the detector is at the optimum level for the Chirascan electronics Tick the Absorbance check box if you wish to simultaneously record an absorbance spectrum of the sample recommended but note that a background must be acquired before the absorbance can be ob
78. ctrometers Absorbance is normally calculated according to T A log SAMPLE 6 6 Treger IS the light transmission with no sample only solvent in the observation cell this is often referred to as 100 transmission or Tioo Tsamrre IS the transmission with sample in place For an absorbance spectrum first the solvent transmission is scanned the background followed by the sample to yield corresponding reference and sample transmissions at each wavelength The absorbance spectrum is then simply calculated as the log ratio for each data pair It is essential for this calculation to be valid that the two separate measurements are taken under identical conditions of detector gain constant photomultiplier high voltage or LAAPD amplifier gain stage However this constraint does not apply to CD backgrounds and spectra because CD is a Self referencing measurement the AC DC ratio is available from a single scan The means the detector gain can be automatically adjusted on the fly to optimize the signal levels and allow the most accurate and low noise CD measurements whether backgrounds or spectra to be made This is done as the wavelength range is scanned and involves continuously adjusting the high voltage on the PMT or the LAAPD detector gain to deliver the optimum photometric DC voltage via the electronics to the analog to digital convertors This high voltage or gain control to maintain the photometric DC voltage is referred to as
79. cularDichrots A Transmission Absorbance Voltage Figure 5 7 example menu of properties that can be plotted as y axis Document 4207Q108C03 01 Page 90 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences Se EE Oa Chirascan Series User Manual When run in CD mode five properties are recorded with every measurement made on the Chirascan These are e Circular dichroism e HT i e HV detector high voltage e Voltage a record of the actual detector DC voltage achieved e Temperature at which the measurement was taken e Count the number of 25 us samples taken at each step in the scan e In addition the Absorbance can be plotted if it was selected when setting the experimental conditions and a background has been run Figure 5 8 and the Fluorescence can be plotted if it was selected and a fluorescence detector was fitted see the Fluorescence Accessories User Manual G C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0004 dsa Absorban C E File View Trace Math Scale Window Help EAL Ce CD CED CS 2 0 U C q fe hee O m fe lt 210 220 230 VVavelength nm Ready NUM K Figure 5 8 absorbance spectra for water red and lysozyme in water relative to N2 5 3 Manipulating Fitting and Converting data 5 3 1 Trace Manipulation The Trace Manipulation dialog box Figure 5 9 can be opened by clic
80. d Ultrasensitive Spectroscopy for the Life Sciences ea oe eee 33 Chirascan Series User Manual 5 9 CD background subtraction theory Residual birefringence in the photocathode of the detector and the sample cuvette means that a non zero CD background is always present and that its contribution will add to that of the sample The systematic error introduced by residual birefringence in the system can be legitimately corrected by measuring it using a blank and then subtracting the background from the total CD spectrum of residual birefringence plus sample The true CD spectrum of the sample will then be revealed However random noise exists on any measured background or spectrum and such random noise can create problems for the unwary If a background is measured and then subtracted from a series of spectra which are subsequently averaged the noise in the result will be overly influenced by the noise in the background Consider Ox y OX2 y 0X3 y OX4 Y OXn Y where x represents the random noise deviation from the underlying spectrum and y represents the random deviation from a single background Taking the average of the deviations 1 _ HSan Ny we N i l As N tends to infinity x tends to zero and the noise in the background subtracted averaged spectrum becomes the noise in the background itself Of course if a separate background were to be measured for every spectrum this problem would
81. d traces Average All Traces calculates and plots the average of all the traces in the display The original traces are removed Removed traces can be recovered using the Trace Manipulation dialog box Section 5 3 1 Simple Math opens a dialog box that enables you to add or subtract a constant to a trace or multiply or divide a trace by a constant It can be useful for offsetting one trace from another for display purposes or for normalizing spectra for easier comparison Smooth opens a window for applying a smoothing algorithm to raw data It allows you to optimize the smoothing before accepting it Convert opens the Convert CD units dialog box Section 5 3 3 For more information on the Fitting Smooth and Convert functions see Section 5 3 5 8 5 The Scale menu Applied Photophysics Chirascan Examples Lysozyme Spe Math Scale Window Help Set Plot Limits Shift P Reset Zoom Shift Ctrl Z c lsa i On line Auto scale Shift O X Logarithmic Shifts L l Figure 5 38 the Scale menu On the Scale menu Figure 5 38 Set Plot limits calls up a dialog box that enables you to set the limits for the x and y axes Reset Zoom resets the scale to the original display size Double left clicking anywhere in display window will achieve the same thing X Logarithmic will change the x axis to a logarithmic scale This feature can be useful for kinetic data Document 4207Q108C03 01 Page 108 of 133 Applied hoto Ultra
82. deevbilenemucuitin 72 Figure 4 23 Ne GOMMNOUPATION TS MU seinoria niii a iia aaa iaaa 73 Figure 4 24 the Select SHU dialog DOX cccccsessccccccssececcceessececccueusececcseaueeeesseuaeeeeseeaueeeeessaageeeeessaseeeesssanseeess 73 Figur 4 25 the Help MENU sicratcee see snese acters toneasiatisisadiowedsattotin ain I aisa dal sinudin dx EAEE RRES Ee tin EAE RENEE EKRENE ERAKI 73 Figure 4 26 the Device WINKOW cccccscccccssseccceeseecceeseeccuaeeecseuscecceuseesssaueeessaeeeeseaseeessaueeessaseeessauseeessaseessiageeesas 74 Figure 4 27 the Monochromator Window ccccsecccccseeceecaeeeeecneeeeeesaeseeesseeeeessaeeeeeeeaseeeseeeeeesseneeessageeeeseaseessseeeeneas 15 Figure 4 28 the Attenuator Shutter Control WINKOW cccccccceccseeeeeeeceeeeeeeeeesaeeeceeeeseeeeceeeessaeeeeeessaaeeeeeessaaeeeees 76 Figure 4 29 the CD DC photometric Channel WINGOW ccccccccseeeeeeeeeaeeeeeeeeeeeeeeeeesseeeeeeeesseaeeeeeessaaeeeeeessaaseeees 77 Figure 4 30 the Viewer TAD vsserciveicxsngcitevaandatesneataieatentalanseatdiuriemnsientenenbeniaviaboniiuntiesientsianbindubeniaetiluskendiertensaiantiaaulesies 79 Figure 4 31 the CD tab ccccccccccssssececcceeseeeeccseueceeccseaeceecssaaeceecssaaseeeesseaaseeeessuageeeecssuaseeeeessaugeceesssaseeeesssaaaeeees 79 Figure 4 32 the Absorbance taD rr ruoisier aeaa E EREE ESE ENEA TES E EE 80 Figure 4 33 the Fluorescence tab cccccccseeeeeeceeeeeeceeeeeecaeeeeesee
83. dling Unit KSHU O A2000 Rapid Kinetics Accessory Set as default SHU Figure 4 24 the Select SHU dialog box Access Control opens a panel showing the names of members of security groups with access to the ProData software Preferences opens the Preferences dialog box in which personal preferences can be set A full description is given in Section 5 7 4 4 12 4 The Help menu Pro Data Chirascan EMULATION MODE Help Topics About Chirascan Figure 4 25 the Help menu The Help menu Figure 4 25 presents two items Document 4207Q108C03 01 Page 73 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences EEEO Chirascan Series User Manual e Help Topics launches the help manual You can browse the contents and search for specific words and phrases e About Chirascan opens a window giving the version number of the Chirascan software 4 13 The Device window 4 13 1 General dit Device Window Motor Electronics System Reset Calibrate ero Uifsets Service Password Hep Figure 4 26 the Device window Select Devices from the View drop down menu to reveal the Device Window Figure 4 26 Many of the icons require a service password or are inactive and their action is not described here Others are only active when a particular accessory is installed and the user is referred to the relevant User Manual The action of the remaining icons is desc
84. e 2 7 operating conditions for the spectrum in Figure 2 26 Page 43 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences 2 9 5 Vitamin B12 Chirascan Series User Manual The CD spectrum of vitamin B12 plotted as CD mdeg against wavelength nm is shown in Figure 2 27 The operating conditions are given in Table 2 8 Scan range r C Program Files x86 Applied Photophysics Chirascan Examples Vitamin B12 dsa Circular Dichroism A File View Trace Math Scale Window Help Taaa MEHEA EE Circular Dichroism mdeg 350 400 450 VVavelength nm Figure 2 27 CD spectrum of vitamin B12 Spectral bandwidth 1 nm Step size 1 nm Pathlength 1mm Concentration 0 2 mg ml Solvent Water Time per point 2 seconds Total No flow 5 L min Document 4207Q108C03 01 Table 2 8 operating conditions for the spectrum in Figure 2 27 600 to 200 nm Page 44 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences be ea rrr na 2 ae Chirascan Series User Manual 2 9 6 Tris ethylenediamine cobalt chloride The CD spectrum of tris ethylene diamine cobalt chloride plotted as CD mdeg against wavelength nm is shown in Figure 2 28 The operating conditions are given in Table 2 9 m C Program Files x86 Applied Photophysics Chirascan Examples Tris ethylenediamine cobalt dsa Circular Dichroism o o A File View Trace Math
85. e CD spectrum of cytochrome C plotted as CD mdeg against wavelength nm is shown in Figure 2 25 The operating conditions are given in Table 2 6 Document 4207Q108C03 01 r C Program Files x86 Applied Photophysics Chirascan Examples Cytochrome C dsa Circular Dichroism A File View Trace Math Scale Window Help aSaanw anga BE Circular Dichroism mdeg 210 220 230 VVavelength nm 200 Figure 2 25 CD spectrum of cytochrome C Scan range 260 to 175 nm Spectral bandwidth 1 nm Step size 0 5 nm Pathlength 0 1 mm Concentration 1 mg ml Solvent Water Time per point 3 seconds Total No flow 5 L min Table 2 6 operating conditions for the spectrum in Figure 2 25 Page 42 of 133 AppliedP hotophysics Ultrasensitive Spectroscopy for the Life Sciences 2 9 4 Lysozyme Chirascan Series User Manual The CD spectrum of lysozyme plotted as CD mdeg against wavelength nm is shown in Figure 2 26 The operating conditions are given in Table 2 7 260 to 185 nm Spectral bandwidth 2mm Spectral bandwidth Pathlength Document 4207Q108C03 01 r C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme dsa Circular Dichroism A File View Trace Math Scale Window Help TS aE 2 BE Circular Dichroism mdeg 210 220 230 VVavelength nm Figure 2 26 CD spectrum of lysozyme Tabl
86. e data storage on disk Since the Datastore drives the acquisition a Datastore filed on disk can itself be trivially used to initiate a new and identical acquisition by dragging it onto the spectrometer control panel All the settings derived from the Datastore are automatically transferred to the control panel configuration Data visualisation and management is carried out with the Pro Data Viewer program Client server architecture means this can operate either on the host computer alongside the control panel or on a remote networked computer This allows experiments in progress to be monitored remotely from the instrument at another location Document 4207Q108C03 01 Page 124 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual The data is transferred to Pro Data Viewer automatically during acquisition so that the experimental measurement appears in real time On completion the new data can be saved on the PC running the linked viewer though a backup is always stored on the host PC as well Once data are collected Pro Data Viewer can be operated independently of the control panel software for navigating amongst data files and performing a range of visualisation and data processing operations see Chapter 4 A range of detailed device windows can be accessed on the host PC for diagnostic purposes These are not usually required during normal operation and in some cases are password
87. e manipulated traces is contained in the History of that Datastore an example of which is shown in Figure 2 18 The file history can be viewed on the View menu on Pro Data Viewer History Time Stamp Fri 4ug 26 16 18 40 2011C Program Files x86 4pplied Photophysics Chirascan Examples Lyso Attributes Time Stamp Thu Nov 24 15 31 36 2005 File ID Is CFR Compliant false Original unaltered data Time Stamp Fri 4ug 26 16 18 40 2011C Program Files x86 4pplied Photophysics Chirascan Examples Lyso Attributes Time Stamp Thu Nov 24 15 39 16 2005 File ID Is CFR Compliant false Original unaltered data mW Time Stamp Fri 4ug 26 16 18 42 2011C Program Files x86 4pplied Photophysics Chirascan Examples Lyso Attributes Time Stamp Thu Nov 24 15 42 33 2005 File ID ls CFR Compliant false Original unaltered data Time Stamp Fri ug 26 16 18 43 2011C Program Files x86 Applied Photophysics Chirascan E xamples Lyso Attributes Time Stamp Thu Nov 24 15 45 49 2005 WW p Figure 2 18 the Datastore History 2 6 2 Exporting data from Pro Data Viewer Right click on a filename in Pro Data viewer launchpad to display the File manipulation pop up menu shown in Figure 2 19 m C Program Files 286 4opled Photophysics ChirascansE sam Lyso 0_ 5mm PLOOO4 dea Lyeo 0_5mm PLOOOS dea Open Browse with Explorer
88. e same for both CD background and CD spectrum For example you might set Low to 180 nm and High to 260 nm for a typical protein Set the wavelength step Document 4207Q108C03 01 Page 26 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE ee eee Os Chirascan Series User Manual size in Step typically 1 nm or less and click Set To the left of the monochromator wavelength bar set the required bandwidth Typically a bandwidth of 1 nm is suitable for protein CD spectroscopy Sampling Time per point a 0 5000 Approximate scan time 73 seconds Current Time per point s 0 5000 Enable Adie aoina Ady Figure 2 7 Sampling panel in the Pro Data interface On the Sampling panel Figure 2 7 set the time per point to the desired value An approximate scan time is shown which is equal to the product of the time per point and the scan range divided by the step size In general the longer the scan time the better the data quality but the chosen scan time will be a compromise between data quality and experimental duration Once the experimental details have been set with the blank in place in the sample holder go to the Signal panel and click Background A CD background will now be measured the display and data manipulation program Pro Data viewer will automatically start and the data will be displayed the data are stored as a file called Backgroundxxxx dsx unless a different seed name has been specified see later
89. ecccaaseececcceaueececceeauseeeeseeauseeeesseaueeeesseeageeeessseageeeesssaaaes 63 4 4 The Monochromator panel ssianicesciwerensudenientavenssaatbvsdawiatwndenistewiantedenkeaialusiantaiesdsadubensaniabesiecdsiusieutatentienauienidas 64 rE I G2 gs e E E ee E E T A A E ee A E E E ee T 64 4 4 2 Advanced mono setup discrete wavelengths cccccsseccccssececeeecccseccceeusceseueeceseuecesseeeessueeesseueeeesaneeess 65 4 4 3 Advanced MONO setup SKIP SCANS cccsesscccsssecccescccseecccseusccssuecccseueecssaueceseaeseesauecesseuseessueeeseeeessaeeess 65 AS Ine Tiggerand LIMeDaSe Panels mcnsnniaiiciorenentanseannciasernimdmcimnieeiueusomteioremarsimmmasminmenteumaleaeds 66 4 6 The Sampling panel i ssciet coviexicwsncdousbacs qrcqesandnneesatiencetitsexecetiovepace tes dug vaPesnabadeensad Gosarncetncdeacd nedeeea sesencebenqnexeseres 66 BoP Vie COU CICS aS harige a A EE ness 68 4 8 The Progress and Status OAM els acicctcnnieasaeccduadencaatinas sath necasaeedenneadivagpccsedanssicad badspsanedecssadeasedeaweaeasecdaddanetwoene 68 4 9 The Temperature Control Unit panel cc cceccccccceeeeeeeeseeeeeeeeesseeeeseeeeeeeeseeeeseeeeeeeesseseaseeeeeeessesaaaeeeeeeess 69 4 10 The Sample Handling Unit panel ESHU panel ec ccccccccceseeeeeeeeeeeeeeeeeeeeeeeeesseeeeeeeeeeeeesssaaaeeeeeeees 69 1 612 621 gl 620 9 ee ee ee eee ener 71 4 12 Drop down MENUS ccccccssseeecccceeseeeecceausececccsaueceecssauecessseuusececss
90. ectrometer Control Panel The purpose of Pro Data Chirascan is to control the spectrometer and its accessories and to acquire data It has been written to be versatile enough to cope with a wide variety of experiments while being simple to use It operates under Windows 7 and some earlier versions of Windows The object oriented modular architecture is easily expandable allowing the introduction of new features anticipating future developments The software is launched by clicking on the desktop icon fo The Spectrometer Control Panel of Pro Data Chirascan Figure 4 1 will appear on the screen Pro Data Chirascan EMULATION MODE File View Configuration Help Signal Circular Dichroism 15 1291 mdeq Close Open Ee Options gt AutoS butter C delta A W Absorbance millidegrees Fluorescence Temperature Control Unit Temperature C Settings E AutoPM T Ramp Disabled Baseline Auto Subtraction Sample Handling Unit ESHU Show Calibration Temperature C 22 1 Ti Titration SHer Monochromator Wavelength nm 280 0 50 0 100 0 150 0 200 0 250 0 Bandwidth nm 1 0 i o io on SIO alent yume A MB me a E Set Low 200 0 High 280 0 Step 1 0 Set Sampling Time per point z 0 5000 Z Approximate scan time 73 seconds Curent Time per point s 9 5000 Enable Adaptive Sampling Sequencer Repeats 2 Trigg
91. ed from the spectrometer for transportation or to install the CS SF stopped flow accessory 25 pin female D Connector Fluorescence detector port Temperature probe Purge gas inlet connector bank Locking ring Circulating fluid connectors CD detector port Support plate Locating peg Lid open sensor Figure 3 6 the interior of the spectrometer Sample Handling Unit The interior of the ESHU is shown viewed from the front with the lid raised in Figure 3 6 the aperture for light inlet from the monochromator is to the left opposite the CD detector port There are two detector ports on the ESHU to the right and rear Normally the right port is used for CD and the rear port for fluorescence The purge gas inlet Is to the left rear upper the circulating fluid connectors to the right rear lower and the electrical and additional temperature probe connectors are above the circulating fluid connectors On the right within the ESHU is a lid open sensor when the lid is raised no experiment can be started and any that are in progress will be paused Document 4207Q108C03 01 Page 54 of 133 AppliedPhotoph Ultrasensitive Spectroscopy for the Life Sciences SE L Pedra E AE ER EA Chirascan Series User Manual Thumbscrews Figure 3 7 the interior of the ESHU viewed from the right one thumbscrew is obscured by the fluorescence detector locking ring The ESHU attaches to the monochromator section of the spectrometer with fou
92. ed trace The smoothed trace and optionally its residual are plotted Section 5 3 Opens the Convert CD units dialog box enabling you to convert from the current CD units to others Section 5 3 3 Opens the Trace Manipulation dialog box Section 5 3 1 Opens the Visible Trace Range dialog box to reduce the number of traces from the same Datastore that are displayed Document 4207Q108C03 01 Page 102 of 133 Applied hotop Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual TR Removes all overlaid traces from the display An overlaid trace is one whose property and dimension e g CD and wavelength are the same as those of traces already in the display but whose wavelength range or step size or bandwidth differs from the originals TH Changes the x axis to logarithmic scale Clicking again on the icon will change the axis back to a linear gcale A logarithmic scale is mainly used when time is plotted as the x axis Resets the trace colours to the first available in the palette This can be useful after trace manipulation if the automatically assigned colours are not suitable e g too pale 5 Launchpad drop down menus 5 7 1 The File menu View Directory Preferences Help New Ctrl N f43dee Open Ctrl 0 Delete Rename Exit Figure 5 27 the File menu The File menu Figure 5 27 presents the usual Windows options for opening and closing files 5 7 2 The View menu
93. eeeeeeaeeesseeeeesaeeeeesseseeessaaeeesseeeessaeeeeesaaseeessaneeneas 80 Figure 4 34 the Data Acguisiton TAD tusiscusiincrovaeutnaasonacteuseenimenehocnriwondsaswsiecuhelatelmauaohmaveduiimanaqencouataionedaahoebunmecn 81 Figure 4 35 the File Names tab cccccccccsseecccesseeeceeseeeceeeeeceescecceueeeseueeessaeeeeseseeessageeessageeessageeeeseuseeessaeeeeses 81 Figure 4 36 the Start p Preferences taD shed siccceen ehadetns dacen qcatdadehes need en Boadcha edness aca iaaa aiaa i aa aed a i 82 Figure 4 37 th CD Calibration dialog DOX ciacia cecteencttannnstadenntnedtaidnnesnnedesndveceshuseaenseadhesiaactedstaccouscpesantarndactoeddpeadeaess 83 Figure 5 1 Pro Data Viewer showing the launchpad left and graphical display right cccecssseeeeeeeeeeeeeees 85 Figure 5 2 the Datastores Details View cccccscccccsssececeeseeeceeececceuseeeseeseeecsagseeeeeaseeessaseeessageeessageeeeseaseesssageeeees 87 Figure 5 3 two files selected in the launchpad and dragged onto the display ccccceceeeeeeeeeeeeeeeeeeeeeeaeeeeees 88 Figure 5 4 CD spectrum of lysozyme with background selected ccccccccseeeeeeeeeeeeeeeeeeeesaeeeeeeeeeeeeseeeeesaeeeeeas 89 Figure 5 5 full display left and zoom using the left mouse button right cee eceeceeeeeecseeeeeeeeeeeeeseaeeeesaeeeeeens 90 Figure 5 6 zoom using wheel alone left Ctrl wheel centre ccccseeceecseeeeeeeeeeeeeeeeseeeseeeeeeese
94. emperature ramp experiments or during titration experiments when waiting for titrant to be added The main purpose of Autoshutter is to minimise the exposure time of a sample to intense radiation 4 4 The Monochromator panel 4 4 1 General Monochramator Wavelength nm 260 0 50 0 100 0 150 0 200 0 250 0 3 0 Bandwidth nm 1 0 Ce eee see ene eam ee eee etre Low 200 0 High 280 0 Step 1 0 I se Adv Set Figure 4 9 the Monochromator panel The Monochromator panel Figure 4 9 provides access to the parameters that control the operation of the Chirascan monochromator Here you set the scan range step size and bandwidth for your experiment you can also move the monochromator to a specific wavelength To drive the monochromator to a specific wavelength use the Wavelength nm edit box and click the Set button below the box to set the bandwidth use the Bandwidth nm edit box and click the same Set button Setting wavelength and bandwidth can be done simultaneously with a single click To set the wavelength range for a scan use the Low and High edit boxes to set the lower and upper wavelength limits use the Step edit box to set the step size all units are nanometres Click on the Set button to the right of these boxes to confirm these parameters Note the Chirascan always scans from high to low wavelength To save the current monochromator parameters as the default ones click on the Adv button which displays the
95. ength list by clicking on Clear list and then select Append to list Now select Single wavelength specify the wavelength in the edit box and click on Generate list Each time you type in a wavelength and click on Generate list the specified single wavelength will be appended to the list When you are satisfied that the list of wavelengths is correct click OK When you click on Start in the Spectrum panel of the interface the monochromator will drive to the selected wavelengths in the order they appear in the list and measure the CD or other chosen property of the sample at those discrete wavelengths 4 4 3 Advanced mono setup skip scans A simple wavelength range can be set in the Monochromator panel and there is no need to use the advanced panel to set such a range see Section 4 4 However if you wish to carry out a more sophisticated scan select Wavelength range in the Monochromator Scan Setup Advanced dialog box Then clear the wavelength list by clicking on Clear list and then select Append to list Specify the Start Stop and Step values for the first wavelength range and click on Generate list A number of entries that correspond to your specification will appear in the list Then enter the Start Stop and Step values for the second range and click on Generate list once more The new values will be appended to the existing values in the list Repeat this cycle until you have completed the skip scan list The total number of entries in the li
96. ent or refocusing Document 4207Q108C03 01 Page 51 of 133 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences Sea SR ee ta Pek or ASE a Sees Chirascan Series User Manual NOTICE The performance of xenon arc lamps shows some deterioration over time It is recommended that the lamp is replaced after 1000 hours of use The spectrometer lamp panel contains a timer showing the number of hours of lamp operation since the last reset Figure 5 4 The counter can be reset using an object such as a 2 mm Allen key hex key It is recommended that the timer is reset when a new lamp is installed If it is intended to swap between lamps for example when operating with or without an CS SF stopped flow unit a record should be kept of the number of hours for which each lamp is in use Details of the lamps available for use with the Chirascan series spectrometers are described in Sections 3 4 2 to 3 4 4 of this manual Figure 3 4 the lamp panel 3 4 2 Standard xenon lamp The standard light source used with the spectrometer is a 150 watt xenon arc lamp The low wavelength radiation produced by this lamp is responsible for the conversion of oxygen to ozone and this lamp should only be used with nitrogen purging This lamp is not suitable for use with the CS SF stopped flow accessory 3 4 3 Mercury xenon lamp A mercury xenon lamp is preferred to the standard xenon lamp for use with the CS SF stopped flow accessory owing t
97. equency will depend upon usage of the instrument but once every six months or so would be appropriate for most laboratories Document 4207Q108C03 01 Page 84 of 133 Applied hnotophy SICS Ultrasensitive Spectroscopy for the Life Sciences beet ee ATII OSD 2 Ss Chirascan Series User Manual 5 PRO DATA VIEWER This Chapter describes the Pro Data Viewer program which is designed to display and manipulate data and to store them securely 5 1 Introduction Pro Data Viewer can be used either online with a client server link between the spectrometer control panel SCP and Pro Data Viewer or off line as a standalone program Pro Data Viewer opens with two windows the launchpad which shows a list of the files in the current folder and the graphics display where data will be plotted Figure 5 1 The status of Pro Data Viewer is shown on the status bar at the foot of the launchpad window where the words Ready Online or Ready Offline will be found E E eee e fg Pro Data Viewer Se se C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0005 dsa Circular D Sr lS O nmr i nI File View Directory Preferences Help A File View Trace Math Scale Window Help Windows Lyso 0_5mm PLOOOS dsa g amp El A M S 69 CD 0 CRD RD C Users My Data v E A agal Sle pplied Photophysics Chirascan ADDUSERS Config Examples J Lysozyme Spectra te Ly
98. equently used in conjunction with the information from the CD DC channel to derive the circular dichroism of the sample 4 3 The Shutter attenuator panel era HY Close B D W AutoShutter Figure 4 8 the Shutter Attenuator panel Document 4207Q108C03 01 Page 63 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual This part of the SCP controls the shutter attenuator mechanism The shutter attenuator can be opened or closed by clicking on the Open and Close buttons The graphic to the right of these buttons represents of the shutter attenuator status black is closed and yellow is open Click on Zero HV to set the HV on the detector to zero With zero voltage on the detector the button will read Restore HV click on it to restore the high voltage to its previous value Use this before disconnecting or reconfiguring detectors AutoShutter automatically opens and closes the shutter during instrument operation If the AutoShutter check box is not ticked then the Chirascan will not open the shutter at the start of a scan or close it at the end of a scan However if the shutter is closed at the start of a scan the user will be presented with a warning message AutoShutter is selected by default This provides automatic opening of the shutter at the start of a scan and closes it at the end It also closes the shutter while waiting for the temperature to stabilize during t
99. ered Spectrum 0 Kinetics Acquire Fause Timed intervale 01 ms Spectra Kinetics Progress and Status Ready For Help press F1 Figure 4 1 the Spectrometer Control Panel The SCP enables you to provide instructions to the spectrometer so that it carries out an experiment or a series of experiments according to your requirements The interface is supplemented by drop down menus for the management of files or the launching of associated programs Shortcuts to the most commonly used items from the drop down menus are provided by the toolbar icons Document 4207Q108C03 01 Page 58 of 133 Applied hotophysic S Ultrasensitive Spectroscopy for the Life Sciences INETI ANEETA as Chirascan Series User Manual 4 2 The Signal panel 4 2 1 General i 5 1291 mdeg Linear Dichroizm A Absorbance WOrescence Fluorescence Polarisati E Fluorescence Optical Rotation AutoFhd Photodiode Array Voltage Auto Subtraction Show Figure 4 2 the Signal panel The Signal panel Figure 4 2 is used to select the mode of data collection Unless other accessories are fitted four options can be selected from the drop down menu Circular Dichroism Absorbance Fluorescence and Voltage The panel changes appearance to reflect the mode of data collection The panel for fluorescence is described in the Fluorescence Accessories User Manual those for the other three options are described below Common to all four is an oscillos
100. ers The Trace i Trace Manipulation Removed Traces Visible Traces Select All Lyso 0_5mm PLOOO4 dsa Smooth s 0 Lyso 0_5mm PLOOOS dsa Smooth r 0 Lyso 05mm PLOOOB dsa Lyso 0 5mm PLOOO dsa Lyso 05mm PLOOOS dsa Lyso 05mm PLOOOS dsa Average 0 Average 1 Subtracted 0 Deselect All Set Baseline Remove Remove Others Thin Data Average Simple Maths Smooth wo a a co oy oO T ay Convert Figure 5 12 Trace manipulation dialog box after spectrum manipulation Click OK to close the dialog box The result of the above manipulation is shown in Figure 5 13 with the smoothed and background corrected spectrum of lysozyme and the smoothing residuals plotted E File View Trace Math Scale Window Help 15 D 0 10 10 E kenna 5 Y O ae E U 0 a pa g 5 5 2 Q 10 180 190 200 210 220 230 gaaei aE E a A e 0 e la A e VVavelength nm B CAProgram Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0004 dsa Circular D C Eh Ets 250 260 Ready Figure 5 13 smoothed and background corrected spectrum of lysozyme red and smoothing residuals blue Note that even after closing the Trace Manipulation dialog box any removed traces are remembered Should you wish to carry out further manipulation simply re open the dialog and all the traces w
101. es PEATE roe Chirascan Series User Manual On shut down it is recommended that only the lamp is extinguished by setting the black rocker switch to the off O position The electronics can be left powered on quite safely Once the lamp is extinguished the nitrogen should be left on for a few minutes while the lamp cools 2 3 Launching the software II On starting the computer among other icons on the desktop you will see two associated with the Chirascan and EN These are named Chirascan and Pro Data Viewer respectively Double click on the Chirascan icon to launch the Pro Data Spectrometer Control Panel SCP Figure 2 4 Pro Data Chirascan EMULATION MODE File View Configuration Help Circular Dichroism 15 1291 mdeg Close l Open Ee E Auto butter Options delta A Absorbance millidegrees E Fluorescence Temperature Control Unit ae Temperature C Settings AutoPM T Ramp Disabled Baseline Auto Subtraction Sample Handling Unit ESHU Show Calibration Temperature C 22 1 T1 r Titration SHer Monochromator Wavelength nm 280 0 50 0 100 0 150 0 200 0 E andwidth nm 1 Oo I I I I I I I I I I I I I I I I Set Low 200 0 High 260 0 Step 1 0 Sampling Time per paoint 2 0 5000 Approximate scar time 73 seconds Curent Time per point sk 9 5000 E Enable Adaptive Sampling Sequencer Repeats 2
102. ff the detector reverse bias voltage to reduce the internal gain 6 1 4 Control and acquisition electronics When the modulated light beam strikes the CD detector it is converted into a photocurrent proportional to the incident light flux Since the beam polarisation is circularly modulated at 50 kHz a differential transmission of the two states as would be caused by CD in the sample results in a 50 KHz AC component superimposed on a background steady state DC component The CD is calculated from the ratio of the AC and DC components since CD in terms of AA is given by 2 3 AA A Ar Ir I Ur Lt Vac Voc 6 1 where A is the absorbance of the circularly polarized light and 7 is intensity the subscripts L and R referring to left and right respectively V4cis the AC voltage and Vpc the DC voltage The CD may also be expressed in millidegrees the unit of ellipticity The two units can be converted according to the formula 0 millidegrees 32 982 x AA 6 2 The system electronics communicate via a proprietary digital backplane that can support several acquisition and control modules all operating independently and if required simultaneously with no loss of data throughput bandwidth Digital signal processors on all modules are responsible for managing data and decoding and executing commands from the PC Two dual stepper control modules are used to drive the monochromator cam slits and attenuator Addit
103. for example the Workstation PC and temperature control circulator are manufactured by external companies and are supplied with separate User Manuals provided by those companies The operator should be familiar with the contents of those manuals and in particular with the safety and other hazard information contained therein Note that some of the images of the interior of the spectrometer Sample Handling Unit show the CS PCS Single Cell Accessory fitted Users who do not have this accessory should ignore these and other references to the CS PCS in this User Manual The information in this document is subject to change without notice and should not be construed as a commitment by Applied Photo physics who accept no responsibility for errors that may appear herein This document is believed to be complete and accurate at the time of publication and in no event shall Applied Photo physics be held responsible for incidental or consequential damages with or arising from the use of this document COPYRIGHT 2012 APPLIED PHOTOPHYSICS LTD ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF SHALL NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER THE SOFTWARE PROVIDED WITH THE SPECTROMETER PRO DATA CHIRASCAN PRO DATA VIEWER ETC IS THE PROPERTY OF APPLIED PHOTOPHYSICS LTD APL AND IS SUPPLIED UNDER LICENCE APL IS WILLING TO LICENSE THE SOFTWARE ONLY UPON THE CONDITION THAT THE LICENSEE ACCEPTS ALL THE TERMS CONTA
104. future expansion It also allows the same overall architecture to be tailored to different APL products providing a consistent look and feel across the range A feature central to the whole instrument control and acquisition process is the Datastore This object provides a blueprint for experimental data collection and also becomes the data storage object after acquisition In its simplest form it may simply be constructed for a CD spectrum containing the wavelength range required and a time per point parameter etc but in CD titrations it will also contain a dimension describing the concentration range to target or for a temperature ramp the temperature set points for each measurement It will also indicate whether simultaneous fluorescence or absorbance are to be measured The Datastore is designed to be multidimensional and expandable so that if new measurement variables arise it can expand to accommodate them If new devices are added a fluorescence emission monochromator for example this can be integrated quite easily The Spectrometer Control Panel provides the user interface for setting up and executing an experiment It is this part of the software with which the spectrometer user interacts During the set up process a new Datastore object is created which describes the experiment which then drives the acquisition and is used to accommodate the captured data The resulting Datastore object is self contained and forms the basis for th
105. g capabilities However before the Chirascan PC is connected to a network the System Administrator should ensure that appropriate anti virus and optionally anti spyware software is installed and maintained APL cannot be held responsible for failure of the Chirascan PC due to viruses or other malware Installation of 3rd Party Software The Chirascan Pro Data suite is not compatible with most g party firewall programs including Norton McAfee and Sophos firewall programs The Pro Data suite is compatible with Windows built in firewall provided that Chirascan Pro Data software is listed as an exception APL is not aware of any other conflicts with 3rd Party Software and will not be held responsible for failure of the Chirascan software because of any such conflict Upgrading Software and or Hardware The Chirascan software has been developed for use with Microsoft Windows software and has been extensively tested on the PC s supplied by APL APL cannot be held responsible for failure of the Chirascan software if the Chirascan user or system administrator upgrades the PC or the PC s Operating System Document 4207Q108C03 01 Page 9 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eRe SP Chirascan Series User Manual Technical Support and Licensing APL will provide technical support under the terms of the Service Level Agreement for the Chirascan software subject to the Chirascan Software Licence and for the su
106. g heavy water D2O it absorbs less in this region than normal water and is not expensive buy NMR standard D2O from a reputable supplier 4 If you wish to go to 170 nm and these are very difficult experiments to perform you will have to employ a cell with a pathlength lt 0 1 mm and use heavy water 5 Use degassed solvent to prevent bubble formation in the cell and to remove oxygen 6 Ensure the instrument is purged properly with nitrogen The normal flow rates of lows of 1 L min 3L min and 1L min for the lamp monochromator and sample chamber respectively will be perfectly adequate but when working below 180 nm the spectrometer should be purged overnight 7 Itis a good idea to separate the absorbance of the cell plus solvent from that of sample The best way to do this is to measure a background with no cell or sample in place then measure the CD spectrum of a blank cell plus solvent and the total CD spectrum cell plus solvent plus protein Subtracting the blank contribution from the total will give you the CD and absorbance of the protein only The reason for doing this rather than simply using the cell plus solvent as the background is that water or buffer or D2O absorbs very strongly at wavelengths approaching and below 180 nm and it is important that the total absorbance rather than just that of the protein is known so that it can be kept within the reasonable working range If the total absorbance is below about 2
107. ged samples Equation 6 21 To prevent smearing of spectral information the monochromator moves to each wavelength in the scan and the sampling is carried out before the monochromator moves on to the next wavelength The averaged measurement is then a true reflection of the input signal at that wavelength This is in marked contrast to the application of variable time constant filtering during continuous scanning modes on earlier spectrometers Here the smoothed result for a particular wavelength has a decreasing contribution from the signal at earlier wavelengths visited to the point that spectral features can be skewed and attenuated during the process On Chirascan series spectrometers the average sample size is represented by a time per point parameter based on the current sampling period default setting 25 us Because the wavelength drive pauses while the samples are collected the size of the sample is directly responsible for the time spent per point and therefore the overall scan time The user can adjust this parameter depending on the signal to noise required for the new Document 4207Q108C03 01 Page 132 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eee ops een 32 Chirascan Series User Manual spectrum and the time frame desired for the scan However he or she can be confident that the resulting data are always an accurate representation of the original unfiltered data and that the noise reduction is statistically
108. ghout the wavelength range than the PMT used on the standard Chirascan As CD is a photon shot noise limited measurement 2 the increase in quantum efficiency of Chirascan plus provides an approximately two fold signal to noise gain over the Chirascan in the UV region This extends to several order of magnitude in the near IR region effectively extending the wavelength range of the instrument into the near IR The windowless design removes a possible source of birefringence reflective light loss and absorbance losses of light in the far UV Document 4207Q108C03 01 Page 121 of 133 Applied hotop Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual Chirascan Plus Detector Quantum efficiency Standard Photomultiplier Detector 200 400 600 800 1000 Wavelength nm Figure 6 3 quantum efficiency of the LAAPD and PMT detectors The LAAPD detector has a maximum internal gain of 250 This is in comparison to the 1x10 maximum internal gain of the Chirascan PMT To provide a similar level of total gain and fine grained gain control to the Chirascan the Chirascan plus detector has an output stage of multiple cascading low noise programmable gain amplifiers which provide 16 bit control giving an amplification gain from 0 to 4096 in 65536 gain levels Combined with the internal gain of the LAAPD of 200 to 250 this gives a maximum gain of about 1x10 The amplifier gain is recorded and reported by the softwa
109. gnals can be expressed as DCrer J Hrer leer 6 8 DC oye d Pa ae 6 9 where uper and Lsayp are the two photomultiplier gains necessary to obtain DCger and DCsamp from the light transmitted by the reference and sample respectively f represents an appropriate conversion constant dependent on the units of T If however DCrer and DCsamp are the same which is the purpose of the AutoPM function then the following holds Mrep Rer samp samp 6 10 and Der _ samp 6 11 Tsamp E REF therefore A log HL samp 6 12 H REF For a PMT detector Equation 6 7 can be simplified to u K HV 6 13 for a particular detector where og 6 14 n 1 and Z kn 6 15 therefore A loo KEV same 6 16 K AV pre Re arranging this yields A Z log sar 6 17 HV ip Z is the only constant photomultiplier dependent term remaining and can be obtained easily by a simple calibration step For a given detector and dynode configuration it is a dimensionless quantity between 5 and 6 Document 4207Q108C03 01 Page 127 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual Therefore under conditions where the same target DC level is achieved using the AutoPM function Equation 6 12 LAAPD or Equation 6 17 PMT can be used to accurately calculate the absorbance from the detector high voltage used for the reference and sample measurements However in practice the AutoPM fu
110. hat appears on the Background button during a measurement it can also be Paused and Resumed using the buttons in the Sequencer panel Section 4 7 At the end of the measurement the background will be stored as Backgroundxxxx dsx unless a different seed name has been Document 4207Q108C03 01 Page 60 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eee ops een 32 Chirascan Series User Manual specified View gt Preferences gt Seed names toolbar fee If the filename already exists a Save As panel will open in the current working directory and the data can be saved with a different name or in a different location The Auto Subtraction check box becomes active at the conclusion of a background scan tick the box if you wish to subtract the background automatically from subsequently measured CD spectra Show is used to reinstate a previously measured CD background It calls up the Quickview CD Background dialog box that enables you to load any background file Figure 4 5 QuickView CD Background s X Axis Humber of points O nm Ta O nm Recall Last Load Save Fins Smooth OF Cancel Figure 4 5 the Quickview CD background dialog box Click on Recall Last to reinstate the most recently measured background click on Load to browse for other background files The chosen file will appear in the preview window The Smooth button will smooth the trace which is useful if you wish to
111. he ESHU ee cceeccceeeceeeeeeeeeeeaeeeeeeessaaeseeesesaaeeeees 51 Figure 3 4 the lamp panel cccceccccccseeecceeeseeeceeeeeeeeaeceeesegeeeesaeeeeeseaeeeesseaeeeesaaseeeeaaeeeessaueeessaeeessageeeeseueeesssaneeseas 52 PIQUE 3 5 lamp housing TITINGS ssascieis civeiesisesaceadgen cana cersawsneivierssdiuarduntoiassmnaguaaesunivssentiaauneed daassrearanaauaibcoendqneteudeien 53 Figure 3 6 the interior of the spectrometer Sample Handling UNnit cccccseeeeeeeseeeeeeeeeeeeeaeeseeeseeeeeseeaseeesaeeeeneas 54 Figure 3 7 the interior of the ESHU viewed from the right one thumbscrew is obscured by the fluorescence detector TO CITING TING aroi iani e iia edi e eea aa aao oe ieia i ai D 55 Figure 3 8 detector fitted with WINdOW COVED cccccccsseeeceeeceeeeeeeeeuaeseeeeeesaeseeeeeseaeseeeesseeeeeeessaaeeeeeessaaeeeeesssaaeeeees 56 Figure 3 9 the CD detector in position in the ESHU 2 0 ccecccccccseeeeeeeeeeeeeeeeeeeeeeeceeeeseeeeeeeeesaaeeseeesaaaeeeeeessaaeeeees 56 Figure 3 10 the Monochromator dialog DOX cccccccseeeecceeseeeeneeeeecaeeeeesaeeeeeeseaeeeeseaseeesseeeeesseseessageeeeseeeeeesaeneeneas 57 Figure 4 1 the Spectrometer Control Panel cccccsseseccccceeseeeeeeeaeeeeeeeeeeeueeeeeeeeeeeeeeeseeeeeeseeseeeeeeessaaeeeeeessaeeeees 58 Figure 4 2 the Signal panel iwiientaesesnseeetsicnten iaierinantsiqntanathnenmeoaiteintashunianntlasinmnceumiatnadtatntadeaainnnticedeishemanciieetas athabinnotins 59
112. he LAAPD is operated with a constant detector gain but the detector electronics hasa cascade of programmable amplifiers that boosts the gain to optimize the signal See Chapter 6 for more details The variable gain of the LAAPD detector is made to simulate the change in gain with anode voltage of a PMT Consequently the user control of the Chirascan plus is the same as that of a standard Chirascan as well as being familiar to users of other CD instruments The CD DC Photometric Channel dialog permits you to apply a specific gain to the CD detector The PMT HV slider allows you to choose an arbitrary detector gain with the higher the HV the higher the gain applied The AutoPM button will adjust the detector emulated HV so that the LAAPD amplifier gain produces an Document 4207Q108C03 01 Page 77 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual Output signal that is at the optimum level for the Chirascan plus electronics Click the Apply button to apply the HV to the detector The ZeroHV button will turn the gain to zero as well as turning off the LAAPD bias voltage allowing safe removal of the detector In a CD scan the detector gain and so its emulated HV is adjusted so that at every point in the spectrum the anode current is at its optimum level for the Chirascan electronics If this window were open during a CD scan the PMT HV slider and the associated edit box would show emulated HVs that cha
113. hotop Ultrasensitive Spectroscopy for the Life Sciences Sec E AE E AE ROE 5 ep Chirascan Series User Manual EJ C Program Files xB6 Applied Photophysics Chirascan Examples Lysozyme Spectra lyso 0_Smm PLOOOdsa CicularDichwoism fc SH0ANW a opan E Circular Dichroism mdeg 10 180 190 200 210 220 230 240 250 260 VVavelength nm Figure 2 14 Raw data with dialog box called up The first three files are backgrounds the second set of three are the lysozyme spectra Select the first group of three files and click Average Select the second group of three files and click Average Two files named AVERAGE xxxxxxxxxx with the identifying numbers taken from the computer timer will be added to the Visible Traces list and plotted in the display The first of these will be the average of the backgrounds and the second the average of the lysozyme spectra Select the first AVERAGE file and click Set Background Select the second AVERAGE file and click Subtract Background The file SUBTRACTED background subtracted spectrum will be added to the Visible traces list and plotted in the display Select the SUBTRACTED file and click on Smooth The Smoothing dialog box will appear Figure 2 15 Document 4207Q108C03 01 Page 32 of 133 AppliedPhotoph Ultrasensitive Spectroscopy for the Life Sciences ee F Smoothing Savitsky Golay Window Sge V Save Residual On Graph 5
114. ich 12 degrees of smoothing were used on the same data set are shown in Figure 4 8 The structure in the residuals that arises when 12 degrees of smoothing were used indicates that the spectrum has been oversmoothed When you are satisfied with the smoothing process click OK Click Unset Background Select Smooth s dialog box will appear as in Figure 2 16 Document 4207Q108C03 01 and Smooth r and click Remove Others The Trace manipulation Page 33 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences OPP eee ee eee 7 Trace Manipulation Removed Traces Lyso 05mm PLOOO4 dsa Lyso 05mm PLOOOS dsa Lyso O_5mm PLOOOE dsa Lyso 05mm PLOOO dsa Lyso 05mm PLOOOS dsa Lyso 0_5mm PLOOOS dsa Average 0 Average 1 Subtracted 0 Figure 2 16 Trace manipulation dialog box after spectrum manipulation Visible Traces Select All Smooth s 0 Smooth r 0 Deselect All Set Baseline Remove Remove Others Thin Data Average Simple Maths Smooth eal wo g baa 75 gt o a if T Convert Chirascan Series User Manual Click OK to close the dialog box The result of the above manipulation is shown in Figure 2 17 with the smoothed and background corrected spectrum of lysozyme and the smoothing residuals plotted r Saas _ a C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra
115. ill be listed whether visible or removed enabling you to carry on where you left off Document 4207Q108C03 01 Page 94 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Pe ne ne ea E EAT Chirascan Series User Manual 5 3 2 Curve Fitting Select the curve you want to fit and open the Curve Fitting dialog box using by choosing Fitting on the Math menu Section 5 8 4 E on the graphical display toolbar Section 5 6 2 or the Fit button on the Trace Manipulation dialog box Section 5 3 1 arm Fitting Fitting Parameters Preview Single Exponential v Q Q Oe Q OK a exp kx c Fit Range 0 001 1 Set Reset Paramete Yalue Estimated Error Lock 1 034593 0 1084845 a 344 1127 0 4600353 k 8 370788 0 01895376 0 00 0 20 0 40 0 60 0 80 1 00 Time s n 12 11 3 Estimate Plot Evaluation gt gt E 0 02 x Max Iterations 20 Iterations 12 16 0 00 0 20 0 40 0 60 0 80 1 00 Reduced jenna Marquardt 10 variance number Time s vJ Include Residuals in Final Plot Figure 5 14 hydrolysis of glucuronolactone with potassium hydroxide A selected function is fitted to the data using a Marquardt Levenberg algorithm Select the required function form the dropdown list on the Fitting Parameters panel and enter the range over which the data are to be fitted on the Fit Range panel then click Set to update If you are unsure about
116. in this Section in your default working directory The Auto Subtraction check box becomes active at the conclusion of a background scan if you wish to subtract the background automatically from subsequently measured CD spectra tick the Auto Subtraction check box Note that if Pro Data viewer has been launched from the desktop independently of Pro Data itself by clicking on its desktop icon then Pro Data will not know of the existence of the Viewer which is a separate program and Pro Data viewer will not respond to the start of an experiment in the normal way To make the connection between the programs go to Preferences gt Go online in Pro Data Viewer and select Local in the dialog box In general when working at the instrument it is recommended that you launch Pro Data Viewer from Pro Data Chirascan using the Q icon in the toolbar This will ensure that local communication is established that Pro Data viewer will work correctly as a real time display and that the data will be saved to a Datastore in the current working directory Document 4207Q108C03 01 Page 27 of 133 AppliedPhotophysix Ultrasensitive Spectroscopy for the Life Sciences E E es Pedro AE OER EAE Chirascan Series User Manual A File View Trace Math Scale Window Help CEEL L Cod 8 eA CBD Lyso 0_5mm PL0004 dsa Circular Dichroism mdeg 220 230 240 250 VVavelength nm Figure 2 8
117. inter with Ctrl or Shift keys and drag them onto the graphical display Figure 5 3 e To display a file in a new window double click on the filename or choose Open from the File menu Section 5 7 1 e To remove a spectrum from the display select the trace Section 5 2 2 right click and choose Remove Selected Trace or use the Trace manipulation dialog box Section 5 3 1 Document 4207Q108C03 01 Page 87 of 133 Applied hotophysix Ultrasensitive Spectroscopy for the Life Sciences SR a AE ER OD Chirascan Series User Manual fz Pro Data Viewer oj Za C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0008 dsa Circular D o 0 s File View Directory Preferences Help A File View Trace Math Scale Window Help Windows Lyso 0_ Smm PLOOOS dsa J E amp E 4 2 68900A e C Program Files x86 Lysozyme Spectra ma E A agaga El C Program Files x86 4pplied Photophysic Lyso 0_5mm PLO007 dsa EA Lyso 05mm PLOOO4 dsa Lyso 0_5mm PLOOO5 dsa 15 Lyso 0_5mm PLOOOS dsa Lyso 0_5mm PLOOOE dsa Lyso 05mm PLOOO dsa Lyso 05mm PLOOO8 dsa Lyso 05mm PLOOOS dsa A D 10 v l E 5 a 0 ba T 9 D QO amp a D 5 ai iO 2 files selected a 10 180 190 200 210 220 230 240 250 260 VVavelength nm Ready On line Ready NUM Figure 5 3 two files selected in the launchpad and dragged onto the display 5 2 2 Selecting a spectrum in the display
118. ional modules can be fitted to allow simultaneous fluorescence acquisition or the control of sample handling accessories such as stirrers Any additional signal acquisition channels will operate in parallel with existing ones so that sampling frequency is not compromised in multi channel configurations The Comms module which is the interface of the electronics to the USB link to the computer also provides several extra inputs for analog temperature probes and general purpose digital I O Vacant backplane slots are available for future expansion All incoming signals CD AC CD DC fluorescence etc are digitised at a minimum sampling period of 10 us 100 KHz Signals are 1 kHz bandwidth limited to prevent noise aliasing but are not otherwise filtered in the analog domain This is to prevent the risk of irreversible distortion of spectral features which can arise by the incorrect selection of such smoothing filters when the raw input signal is changing too quickly The modular design of the electronics means only required features need be installed and any faults that develop are localised and can be easily repaired by substitution of the appropriate module All modules have a built in Document 4207Q108C03 01 Page 123 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE ee eee Chirascan Series User Manual self test capability which communicates any operational problem to the user and flash memory is used to allow all onboard
119. kes out a service contract Please contact APL Technical Support Section for further advice 3 8 Moving the spectrometer 3 8 1 Parking the monochromator NOTICE The monochromator has components than can be damaged if their movement is not restricted when the spectrometer is moved This movement is restricted by parking the monochromator To park the monochromator open the Monochromator dialog box Section 4 13 2 and click the Park mono button Figure 3 10 Monochromator Mono 1 eS Wavelength nm J Bandwidth nm Status MonolD EUMono2 Current Ready action Hi Temperature C 27 1 Calibration File C Program Files 86 Applied Fhotophpsics ChirascansConfigs evices M onoLal cont Calibration temperature 23 00 C Reload calibration Figure 3 10 the Monochromator dialog box 3 8 2 Lifting and moving the spectrometer AA CAUTION The spectrometer weighs about 80 kg 180 pounds and should only be moved manually if enough people are available to support this weight Ndl AA he spectrometer has designated lift points and lifting at the wrong points can result in damage to the instrument Do not attempt to lift or move the spectrometer except under guidance from APL s technical support section Document 4207Q108C03 01 Page 57 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences eee ops een 32 Chirascan Series User Manual 4 PRO DATA CHIRASCAN 4 1 The Sp
120. king Selection dialog on the Trace menu Section 5 8 3 or by clicking on the graphical toolbar Section 5 6 2 The dialog box is an environment in which data traces can be background corrected averaged or otherwise manipulated All operations carried out within this environment are reversible which means that if errors are made they can be quickly and easily corrected The original data are never over written or otherwise destroyed and all operations carried out on data are recorded in the History file on the View menu Section 5 7 2 The results of the manipulations are displayed in real time and can be saved at the end of the session in one of a number of formats using Save current plot on the File menu Section 5 7 1 Document 4207Q108C03 01 Page 91 of 133 Applied hotophysi Ultrasensitive Spectroscopy for the Life Sciences E E ts Nedra AE OER 5 an Chirascan Series User Manual Trace Manipulation Removed Traces Visible Traces Select All Lyso 05mm PLOOO4 dsa Lyso 0_5mm PLOOOS dsa Deselect All Lyso 05mm PLOOOE dsa Lyso O_5mm PLOOO dsa Set Baseline Lyso 05mm PLOOOS dsa Lyso 0_5mm PLOOOS dsa Subtract Baseline l Remove Thin Data Average Simple Maths Smooth Fit Convert Figure 5 9 the Trace Manipulation dialog box The structure of the dialog box is straightforward There are two windo
121. lbar iCONS sscccccccssssccccceseccccauesececceeesecceseeaseceesaaaeeceeseeaeceesaeaecessuaaeeeessugeeeeees 102 5 7 Launchpad drop down MENUS sssrini airn K RRETA OEST ARE AENEAN A ERREAREN ESTARAN ARA Ri EEREN 103 ILATE FE E I VU e E E E A E E E E AE 103 TV VS WV E a E EE A A E E S E E E E E A E E E eaeraneate 103 A MMe Dr OCO A eee a E AE E EA AEE AAT A O EEA EE 104 SILA Ne PrererenceS WOW eicere aa NE T EE EN 104 5 7 5 The Help MEenU s ssn ssssssnssssreresssreresssrerossrreressreresssreressererossereressreresssreresssrerosserreesseteeessreresssrerossereeessrerens 105 5 8 Graphical Display drop down MANUS ccccccccsseseseceeceeccaeeeseeceeeeeesaeaeeeeeeeeeesseeeeeeeeeeessseaaeeceeeeesssaaaeess 106 ose Me VU UM NN tects EN PE NA ete ares et nate A A gta cio teasing E A E A E E E 106 IS LTE VEN TSI o veces ocean ares scene ce on ecto ote E eaten Picton onsen A A 106 38 NE Mace ME Meinir E EE N doe daw uae maeas seokoslas e E 107 POETE MANE eS ace ovaanecubasacearsntaaasinesoseesncsceorsansaneeueuranacaerancseasaeancs 108 58 9 The Scale MENU semreriniunirinre i E a 108 TE EAN NAON E eeoa AN N E E E 109 FS TOPER A sa a E E E E tne ae anaes one winoSeamuto mandi ncntnacdade 109 5 9 CD background subtraction theory ccccccccecccsseseeeceeeeeecaeeeseeceeeeeeeaeeeeeeceeeeeessaeseeeeeeeessaeaaseeeeeeeessaaaaesss 110 5 10 Using CDNN to calculate the secondary structure Of proteinS sssesssssssnnnsessrnrrrr
122. les you to choose between Absorbance and Transmission as the measured variable If the fluorescence accessory is present you will optionally be able to record a simultaneous fluorescence excitation spectrum by ticking the Fluorescence check box For more information on making fluorescence measurements see the Fluorescence Accessories User Manual The Background subpanel is used to measure a reference background relative to which the absorbance of the sample is calculated Selecting Auto HV in Background will optimise the high voltage applied to the detector for every point in the background scan These values are remembered and applied at each corresponding point in the subsequent absorbance scan allowing an accurate absorbance spectrum to be recorded easily Clicking on Background will initiate a background measurement using the experimental parameters defined elsewhere in the interface The measurement can be stopped at any time by clicking Stop which is the label that appears on the Background button during a measurement At the end of the measurement the background will be stored as Backgroundxxxx dsx unless a different seed name has been specified With the background measured insert the sample and click on Acquire in the Sequencer panel to measure an absorbance spectrum Document 4207Q108C03 01 Page 62 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eee an ops een 32 Chirascan Series User Manual 4 2 4 The Signal p
123. line Add Baseline and Subtract Baseline become active once a baseline has been set and respectively add a baseline to a selected trace or subtract a baseline from it Remove Overlaid Traces removes all overlaid traces An overlaid trace is one whose property and dimension e g CD and wavelength are compatible with those of traces already in the display but whose wavelength range or step size or bandwidth is incompatible with those traces already in the display Overlaid traces cannot be manipulated and are always coloured black to differentiate them from active traces Recompute Trace Colours will assign new colours to the traces in the display This is useful if after manipulation the colours of the traces are not satisfactory Reset will undo all operations carried out on the original data and restore the original display Document 4207Q108C03 01 Page 107 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Pa ean Eee Ore Chirascan Series User Manual 5 8 4 The Math menu s x86 Applied Photophysics Chirascan Examples Trace Scale Window Help he Fitting F yo06 d Average P Average All Traces Shift 4 Simple Math Mi Smooth 5 J Convert Shift C Figure 5 37 the Math menu All mathematical functions can be called up using keyboard shortcuts or toolbar icons Fitting calls up the Curve fitting dialog box Average calculates and plots the average of the selecte
124. lp MENU seremarieninteniaine i rnant reenen ianen ENAP i EAI EETA EARNE SENARE ANANE ERREKAN EE ERNARI ENA ATEAN 109 Figure 5 41 multiple lysozyme spectra ce ecccceeeseeeeeeeeeseeeeecesauseeeeeeeasseeeeeseasseeeesseasseeeesseasseeeenseaseeeeenseaseeeeensagses 110 Figure 5 42 smoothing residuals left and their average right cccccecccccssececceeceeceeseeeeeeeeceeseeeeeeseaeeeessaaees 111 Figure 5 43 single background corrected residuals left and their average riQht cccccsseeeeeeeeeeeeeeeeeeeeeees 111 Figure 5 44 single smoothed background corrected residuals left and their average right c ccccseeeees 112 Figure 5 45 the Simple Spectra dialog DOX cccccccccsssssseeeeeeeeeeaeesseeeeeeeeeeeeaaeeeeeeeeseeeaaseeeeeeeessaeagseeeeeeessaaaasees 113 Figure 5 46 the Sample Files folder cccccsseeceeceeeeeeeeeeeeeeeeeeeeeeaaeeceeeeeeeesaeeeaceeeeeeeeseeeaaceeeeeeeessaeaaeaeeeeeessaaaaaeees 113 Figure 3 472 the CD Signal Type dialog DOX secacsececeachcenteccantinc cs cdsteewedraceaxnatnce casdcatveesalncaccdeamantoneteuseeieenaeoietacen 114 Figure 5 48 calculated components of the secondary structure Of IVSOZYME cccccseeeeeeeeeeeeeceeeeeeeaaeeeeeaeaeess 114 Figure 5 49 Lysozyme and background spectra several repeat measurements ccccceeeeeeeeeeeeeeeeeeeeeaeeeeees 115 Figure 5 50 Lysozyme spectrum after averaging background correction and smoothing
125. ls prior to digitization whereas the second two are flexible and under operator control 6 5 2 Electronic filtering Firstly prior to digitization of the analog signals it is important that signals are bandwidth limited to less than half the A D sampling frequency the Nyquist critical frequency to prevent the aliasing of high frequency noise into the frequency spectrum of the measurement This is well known from sampling theory 3 and is essential in any instrument which digitizes analog signals at discrete intervals A 200 us 1 KHz low pass filter on the CD channels AC and DC provides this filtering and also rejects any components of the PEM carrier frequency 50 kHz This fixed time constant is the only pre acquisition filter applied to the analog signal and is not user adjustable 6 5 3 Signal Averaging The digitized signals will still exhibit photon shot noise passed by the electronic filtering stage This noise can often mask spectral features or at least make them difficult to see particularly in demanding regions of the spectrum such as the far UV Filtering of this noise is carried out primarily for cosmetic reasons The primary method of noise filtering over which the user has control is sample averaging This is carried out following digitization but during the acquisition process and simply involves averaging digitized samples together This yields a signal to noise improvement proportional to the square root of the number of avera
126. ltrasensitive Spectroscopy for the Life Sciences iota finest resets erent EA R 5 opens Chirascan Series User Manual 4 14 5 The Data Acquisition tab The conditions on the Data Acquisition tab Figure 4 34 have been established as optimal by Applied Photophysics engineers and they should not be changed except under guidance from ALP Technical Support Preferences Viewer CD Absorbance Fluorescence Data Acquisition File Names Startup Preferences Spectrum Spectra Kinetics Mode Options Per point acquisition delay ms 5 Pre scan delay ms 50 Post scan delay ms Detector protection DAG Calibration V PMU saturation protection Calibrate zero offset errors at Zero HVs before spectrum scan er V Zero HVs on fly back spectrum mode A ADDIY PppIy Figure 4 34 the Data Acquisition tab 4 14 6 The File Names tab Preferences Figure 4 35 the File Names tab On File Names tab Figure 4 35 seed names for spectra and backgrounds can be set In a series of experiments the file names will share the chosen seed name and be differentiated by an automatically incrementing numeric component e g Protein0001 Protein0002 This panel can be accessed directly using the File names icon on the toolbar Section 4 11 Document 4207Q108C03 01 Page 81 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FPR re eh
127. lysozym _ flavodoxin __ myoglobin gapdh PolyGlu _ hemerythrin subtilisin C hemoglobin H Idh Thermolysin tim Lo Lo L File name lysozym Files of type ASCII Two Column Data Files t4 Figure 5 46 the Sample Files folder Document 4207Q108C03 01 Page 113 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eee ops een 32 Chirascan Series User Manual Select one of the sample files and click Open Here the Lysozyme example file Lysozym txt is used The CD Signal type dialog box appears asking the user to select the CD signal type Figure 5 47 CD Signal Type Please indicate type of data units Be carefull Wrong assignment will result in incorrect data renormalization CD Signal Type C Molar Ellipticity C Milli Degrees Figure 5 47 the CD Signal Type dialog box All the examples in the Sample Files folder are converted to Delta Epsilon the differential extinction coefficient in this case further normalised for the number of amino acid residues discussed in Section 5 10 3 Click OK The panel will disappear returning you to the Simple Spectra dialog box Click Deconvolute to calculate the contributions of the various components to the protein secondary structure Figure 5 48 Aa Deconvolution File Edit Help 1E0260 nm 185 260 nm 190 260 nm 195 260 nm 200 260 nm 205 260 rim 210 260 nm AZ o 3893 HBX 31 NIZ F5 o4 TE hes PO P14 To 16 4 16
128. mains outlet is powered on No detector signal Put the rocker switch in the T on ignited oosition and press the red ignite button the closed position shutter No light slits too Check the bandwidth in the narrow or closed Monochromator panel of the Pro Data interface and set the bandwidth to a reasonable value e g 1 nm No HV on the detector In this case light might be reaching the detector but without any high voltage applied to the detector there will be no signal Check the detector cable is correctly connected and then use the Pro Data interface to restore the HV No light sample is Reduce concentration and or opaque pathlength of sample and or buffer No light in far UV Oxygen absorbs light at wavelengths instrument is not purged below 190 nm Ensure that the purge tubes are connected at both ends and that there is nitrogen available and purge the instrument with clean nitrogen Signal is too noisy Bandwidth too narrow Check the bandwidth in the mono section of the Pro Data interface and set it to a reasonable value e g 1 nm sample or buffer is Reduce concentration and highly absorbing pathlength of sample and or buffer Lamp is old If the lamp has been run for more than 1 000 hours its output especially in the far UV will be low Replace lamp Electromagnetic Remove the source of noise from the interference from nearby proximity of the spectrometer equipment Docume
129. meter is digitally averaged to reduce measurement noise The raw signal is bandwidth limited before digitisation at a default frequency on all channels of 40 KHz max 100 kHz The signal to noise s n improvement yielded by this averaging follows a square root relationship to sample size N i e sino VN 6 21 The averaging is carried out in the Windows driver and can be controlled by the operator from the spectrometer control panel The sample size for each average is more usefully expressed as a resulting time per point e g if N 40000 the time per point will be 1 second at a sample period of 25 us The spectrometer moves to and stops at each wavelength in the spectrum to carry out this sample average before proceeding to the next wavelength The overall scan time is given by the following relationship Document 4207Q108C03 01 Page 128 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences pest AE rR E Chirascan Series User Manual Scan time Time per point step time x total wavelength steps The step time is a time overhead comprising a contribution from the time taken for the wavelength drive to move to the next wavelength and a short settling time prior to signal acquisition It only becomes significant during rapid scans with minimal signal averaging Total wavelength steps is also equal to the product of the scan range and scan resolution in steps nm plus 1 CD is a demanding technique due to the commonly very
130. mic quarter wave plate 1 modulates the beam into alternately left and right circularly polarised states Document 4207Q108C03 01 Page 120 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual The modulated beam then passes into the sample handling unit It passes through the sample block cell holder and then on to the photomultiplier PMT or large area avalanche photodiode LAAPD CD detector The standard detector port is in line with the beam but a second port is available at the rear of the housing This may be used to accommodate a detector for simultaneous fluorescence detection or fluorescence detected CD FDCD Two bulkhead access plates are fitted to allow other accessories to be placed in the light tight sample handling unit These include a Peltier temperature controller CS PCS a 4 cell autochanger CS PCM and a titration accessory CS TT as well as devices of the user s own design Removable sample holders are provided as standard with the CS PCS to accommodate a variety of square and circular sample cells Up to four temperature sensors can be monitored and magnetic stirrer support can be added as an option The sample handling unit has been designed to accommodate large specialist cell holders and accessories for example cryostats and electromagnets for magnetic CD MCD experiments However in exceptional circumstances the entire housing can be straightforwardly rem
131. minimum such as the APC Smart Ups SUA 1500i Computer requirements The spectrometer is normally supplied with a computer However some users may wish to use run the instrument from their own PC This should be a Windows 7 Pro ready PC 2 4 GHz 2 GB RAM 2 GB hard drive 1 free PCle slot Nitrogen purge gas Unless used with an non ozone producing lamp the spectrometer requires constant purging with 99 998 oxygen free nitrogen at a pressure of 4 to 6 bar 60 to 90 psi and flow rate of at least 5 litres per minute Fluid circulator unit The spectrometer can be used with or without temperature control If used with the CS PCS Single Cell Temperature Accessory or CS PCM 4 Cell Autochanger Accessory for temperature control a water circulator is required with a minimum pressure of 0 2 bar maximum 1 7 bar 3 to 25 psi A CS JS Recirculating Cooler or a CS B100 Circulator both available from Applied Photophysics is recommended Document 4207Q108C03 01 Page 8 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual GENERAL INFORMATION Computer configuration Your Chirascan series spectrometer is normally supplied with an HP Compaq PC pre configured for use with the instrument However there are some issues that you need to be aware of described below Administrator login No password is set by APL for the Administrator account Customers are advised to set the Administrator
132. moved The design of the spectrometer provides protection against electrical shock by earthing grounding appropriate metal components This protection will be lost unless the power cable is connected to a properly earthed outlet It is the user s responsibility to ensure that a proper earth connection can be made 3 2 1 Mains connections The Chirascan has two electronics units mounted above the monochromator When viewed from the rear of the spectrometer the lamp electronics unit is to the right the monochromator electronics unit to the left Each unit requires a mains electricity supply at the local voltage Figure 3 2 Document 4207Q108C03 01 Page 49 of 133 Applied hotophysi Ultrasensitive Spectroscopy for the Life Sciences SE E AE eee A Chirascan Series User Manual Figure 3 2 mains electricity connections 3 2 2 Module connections In its standard configuration the Chirascan has four modules fitted in its electronics rack labelled USB PEM CONTROL 1 and CONTROL 1 Table 3 1 The two CONTROL 1 modules are distinguished by labelling their release buttons M1 and M2 The USB module has three connection ports the others have two ports Additional modules may be installed for accessories for which see the relevant accessory User Manual Module USB PEM CONTROL CONTROL MODULE MODULE MODULE 1 MODULE 1 Upper port DRIVE DRIVE 1 DRIVE 1 Lower port TEMP PMT Chirascan or DRIVE 2 DRIVE 2 INPUT LAAPD Chirascan plus Rele
133. n empty lamp storage box ready to receive the old lamp Power off the lamp and leave for at least 30 minutes for the lamp to cool down Remove the spectrometer end cover by sliding it to the left off the magnetic catches and clear of the lamp housing Unplug the red and black leads at the back of the lamp power supply Unscrew the two retaining screws on top of the lamp assembly Carefully lift the old lamp out of the housing and place in the storage box securing with the retaining screws Unscrew the retaining screws from the new lamp storage box remove the lamp and carefully insert into the housing Secure the lamp in the housing with the retaining screws Reconnect the red and black leads and replace the spectrometer end cover Purge the lamp housing at 5 litres per minute for at least 10 minutes then reduce the flow to normal operational level Power off the lamp wait for about 10 seconds then strike the lamp and reset the timer on the lamp supply Please return the old lamp assembly to APL for recycling Document 4207Q108C03 01 Page 53 of 133 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences bo iat ee te ie DRED 2 es Chirascan Series User Manual 3 5 The Spectrometer Sample Handling Unit ESHU The spectrometer Equilibrium Sample Handling Unit ESHU is the lidded chamber that mounts to the right of the monochromator unit where it is held in place by four thumbscrews The ESHU can easily be remov
134. n optimised to maximise the signal to noise ratio of the PMT detector of the Chirascan In what follows the description of the algorithm is based on the Chirascan PMT but the Chirascan plus LAAPD has been designed to have a similar relationship between emulated HV gain and photon shot noise and the algorithm works equally effectively By reducing the number of samples reducing the sampling time per point in regions of high light flux a spectrum can be measured in a shorter time Put another way the use of adaptive sampling can lead to better Document 4207Q108C03 01 Page 129 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PEATE roe Chirascan Series User Manual spectra being measured in the same time With careful experimentation using adaptive sampling can produce better CD spectra in a shorter period of time Although the relationships between HV gain and photon noise are quite complex it is also true that for a given detector the observed photon noise amplitude is a direct function of the applied HV and to a large extent independent of other instrumental parameters such as wavelength or bandwidth By using the relationship in Equation 6 6 it is possible at any HV to equalise the noise in averaged data by a suitable adjustment in the sample size i e as the light flux reduces and the HV rises when a doubling of photon noise occurs this can be compensated for by a 4 fold increase in the average sample size This i
135. n the file Figure 2 12 F C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0007 dsa Circular D C B E File View Trace Math Scale Window Help x ECELEHSRBRBSILEeRIKUAS iee e Eee l 7 Circular Dichroism mdeg 210 220 230 VVavelength nm Figure 2 12 CD spectrum of lysozyme raw data Double click the left mouse button anywhere on the graphical display to autoscale the axes such that you see the full soectrum To zoom in to a particular part of the display click and hold the left mouse button whilst moving the cursor to create a box around the area of interest On releasing the left mouse button the newly created box will Document 4207Q108C03 01 Page 30 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PPE re hr eRe OE Chirascan Series User Manual expand to fill the display window To return to the original display simply double click the left mouse button at any time To zoom in or out in both x and y axes simultaneously use the mouse wheel to zoom in or out in the x axis only use Ctrl in conjunction with the mouse wheel to zoom in or out in the y axis use Shift in conjunction with the mouse wheel To view the other properties measured simultaneously with the CD spectrum right click on the y axis label and choose the property from the dropdown list that appears or select New Window on the Window
136. naaten 81 4 14 6 The File NAMES taD ssicsescecsssscscorsvenenessenesssennesssenswessssssessenosesencsesesabesesseedssencsessedenteseconsseheuessensoesssnesevens 81 4 14 7 The Startup Preferences tab nnossennssseeenssserersssrrresssreresserrrossereesssrereessrrresssrerossereeosserresssrereeseeeresseree 82 4 15 Recalibrating tne GAIN ASC AN esns ii a i a iie 82 4A 15 1 Recalibrotine the CD 4 s4sssisssssaunctanssuacavegs soncueussvcosaddaesdsene aE E EE 82 4 15 2 Recalibrating the derived absorbance Chirascan only ccccccccecccesssseecccceeseeeesecceceeeseeeeeeeceeeesauegeeeeees 83 FRODA VENE P erene E E E EE EE EE 85 SE ROO CUIC Ree set A E E E E E ET E E E E E A E E T 85 5 1 LaUnEnIng Pro Data VIEWER ciscicinnavavslasazscasiwcdnvabneseenasenedinninnadncdeen a E E 86 5 1 2 File structure the Datastore saecdenpeen van auceassenceseunteongneeavaneeasaieeocsaeacanenaeyansnn eas eenonesqanianaas ear dnesaevaueaceomatere 86 5 1 3 Viewing experimental conditions ccccsesccccsseccceeccccsseccseueccsaueeecseuseessauecessuecesausseeseascessausessauesessaaees 87 5 2 Displaying and selecting data cccccccccceeseccecceeescececseeeeceeecaeeseceeecsaaseceeeseaaeeeesessaaseeeesseaeeeeessaageeeesssaaess 87 IAL VIS BaD CAll CSPI AY minsen riie EE ENEAN EEEE NES 87 5 2 2 Selecting a spectrum in the Cisplay corssier in ae ea a r S EN E E E A E E EENE ERA 88 5 2 3 Selecting more than ONE SPECTFUM cccccsss
137. nction returns when a signal DC level is within a tolerance window close to the target value This lack of accuracy would introduce errors into the calculation above However this error can be corrected by re introducing a term based on the classical calculation in Equation 6 6 From Equations 6 6 6 8 and 6 9 A log 22 Jog Hsane Crer 6 18 SAMP Hrer DC samp Here we retain the individual DC terms for sample and reference to account for the AutoPM discrepancy For an LAAPD detector this formula allows an accurate absorbance to be calculated when the individual DC signal levels are recorded together with the detector gains For the PMT detector we can derive the following A log XPV sau P Cree 6 19 K AV pee DC gaye and finally REF D Csamp Which allows an accurate absorbance to be calculated when the individual DC signal levels are recorded together with the PMT detector high voltages lt can be seen that in the event of the HV being constant and the AutoPM function is not applied Equation 6 20 simplifies to the classical Absorbance calculation in Equation 6 6 If the AutoPM function is perfect and the target DC signals are the same then only the left hand term applies and the calculation is the same as in Equation 6 17 Any intermediate situation is accommodated through application of the full expression Equation 6 12 APD or Equation 6 20 PMT 6 4 Adaptive sampling 6 4 1 Introduction Incoming signal data on the spectro
138. nd monochromator have slow vent points to allow the internal atmosphere to be displaced by nitrogen which means that once the initial high flow purge is complete there should be no need to repeat the high flow rate purge unless the purge has been interrupted for longer than a weekend If the purge has not been interrupted for too long resuming normal nitrogen flow for 30 minutes is usually sufficient to re establish a fully purged light path 2 2 Powering on Ensure that the instrument is connected to the electricity supply and that it has been purged according to Section 2 1 Put the two black rocker switches System and Lamp on the front panels Figure 2 2 and Figure 2 3 to the power on I position The instrument will automatically check its zero positions you may hear the monochromator motors moving Ignite the lamp by pressing the red Start button on the lamp control panel no sooner than 10 seconds after the lamp has been powered on this is to allow sufficient time for the capacitors to charge up If ignition is successful the translucent panel on the source housing adjacent to the control panel will glow yellow if a xenon lamp is fitted or yellow green if a mercury xenon lamp is fitted if you do not see a yellow or yellow green glow press the red Start button again repeat until ignition is achieved The lamp will require at least 15 minutes to stabilise following ignition Finally ensure that the computer its monitor and printer are
139. ndow Figure 4 28 I Attenuator Attenuator Shutter Control Attenuator Controls Close Open Limit masimum attenuator opening to Y Current attenuator position Maximum open position i marked by selection m Closed Fully Open Figure 4 28 the Attenuator Shutter Control window The attenuator operates both as attenuator and shutter The Open and Close buttons have the same action as those found in the Pro Data interface Section 4 3 and are linked to them changes in one set are replicated in the other The maximum opening can be limited using the slider bar and this may be appropriate in special circumstances for example to reduce damage to samples but in general the attenuator should not be limited in its maximum aperture The status of the aperture is shown on the Current attenuator position slider if the maximum aperture were limited then that would also be shown in the slider position The shutter display on the SCP is linked to this function and therefore the status of the attenuator can be ascertained from there too Document 4207Q108C03 01 Page 76 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences E R are eee Ree Oe Chirascan Series User Manual 4 13 4 The circular dichroism DC channel the CDDC icon displays the CD DC Photometric Channel dialog box Figure 4 29 CD DC Photometric Channel Channel 4 PMT Control Apply Zero Hy AutoPM Ready Module I
140. nenacuy ETAK ANE ANENA OEE AEN EREE eS 103 Figure 5 28 the VIW MENU rirerire aiaa RENER A ETE EEE E a aa EEr AE 103 Figure S29 Me Directory MEMU seeren Enen ne E E EE A E SAARE E REE E ER R 104 Figure 5 30 the Preferences Menu ais osisunronsvduacesaasosicadaiteniausatonvantud manda lontencdibateatetwarcebellesndenabocteupliuntvesisunkeabadusy 104 Figure 5 31 the Connect dialog DOK arts sane satircanaticntinaiss ates omits untadieh oucieasticaanatihnies avignaanicatsknuduarexeank siukeueaasasuant sigiwarvch 105 Figure 5 32 the CONNECTION error MESSAGE ccccccceeneeeseeeeeeeeeeaeeseeeeeeeeeeeeeasaeeeeeeesseeaasseeeeeeseesaaasaeeeesesssaaaasees 105 Figure 5 33 the Help MENU cccceesceeeeeeeeeeeeeeneeseeeeeeesaaseeeeeneaaseeeeeneaaaeeeeeeeaaseeeesseaaaeeeeeseaaseeeenseaseeesensaaaeeeeensagses 105 FOUG 51942 Me TIC ME adiancesmcassrane cassrsacsanmtecd quspseqaqenmunceqiegsecesausgondauessnanaeteeasaaternece Gaumtard ghatameeneonanaaieoactqamecdan 106 Figure 5 39 the View TMC isisisi E E O AEEA EE a E EE 106 ROU S 30 ne ACS MMe HU e A E a E E quaviaiinmane 107 Figure s 37 he Math Meni serria a i aE a ani aa a piana 108 Figure 5 38 the Scale ManU sat settee price enna dit pci reartcee tie wehin ea stadt AEREN EKERN RAA EREEREER AEREA EEEREN ES EAER ENERE 108 Figure 5 39 the Window Menu nes ssanencuscsasearareaanniiarnietanaeakencyseanciontidnanadesededen tanieemdiaanddeen UadncaciaesnehenDodouauenoodseeraniiaacian 109 Figure 540 the He
141. ng by rotating anticlockwise counterclockwise and gently withdraw the detector To protect the detector fit the window cover when the detector is not installed on the spectrometer Figure 3 8 Document 4207Q108C03 01 Page 55 of 133 Applied hotop Ultrasensitive Spectroscopy for the Life Sciences RE ee Ee a Chirascan Series User Manual Figure 3 8 detector fitted with window cover To replace CD the detector perform the above operation in reverse ensuring either that the spectrometer is powered off or that the detector high voltage is set to zero before reconnecting The shoulder of the detector should seat down onto the nitrogen purge ring Figure 3 9 Fluorescence Detector CD Detector Figure 3 9 the CD detector in position in the ESHU For instructions on the installation of the fluorescence detector see the Fluorescence Accessories User Manual Document 4207Q108C03 01 Page 56 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences E eh eae ap Chirascan Series User Manual 3 Maintenance If used with care the Chirascan series spectrometer should provide many years of reliable service eyd Do not allow aggressive or corrosive chemicals to come into contact with the spectrometer clean up any spillages immediately To clean the spectrometer use a soft cloth with a dilute solution of mild detergent in water APL suggest that the instrument is serviced regularly and preferably that the user ta
142. ng diode PEM Photoelastic modulator PMT Photomultiplier tube SCP Spectrometer control panel SHU Sample handling unit HYPERLINKS This document contains hyperlinks between references for example the Contents tables or references to Sections or Figures in the text and sources To follow a link place the cursor over the reference and use CTRL click Hyperlinks in the text are indicated by underlined blue font Document 4207Q108C03 01 Page 11 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences sRSnnes sReatenersetanenss Chirascan Series User Manual CONTENTS USE OO TAG DOC OMEN cae eects acntasena EE A 4 HAZARD AND OTHER INDICATORS canseusntentassnrsaneetondateusivasiaiiacausrreendondtensridanitacinusnniastmeneierataesnimcnoneranes 5 ESSENTIAL SAFETY INFORMATION nsaeaeeenennsrnsrnsrnsrnsrnnrnnrnnrrnrrnrrrrrnsrrnrrnrrnrrnrrnnrnnrnnrurrnnrnnrnnrnnronnonnonnonnenrenrennenrenae 6 INSTALLATION AND OPERATIONAL REQUIREMENT S aansnannoennnsnnnnannnsnnnsnnnonnnrsrrnerrrrnrnnsrrronrrrnrrnenrrrnrrrerreenrrennne 8 GENERAL INFORMATION eurrera n uaa aa aE EAA A TAREE EE E AER AA aAa RAEE EE ERa 9 CY P eset ete E ash E E EEEE O ih tet E tn E E oi dita aia Rea E 11 CONTENTO eei R putecdan eodeedueugawonnesteadaenteedameudiedaasuntsian O R R 12 ROURE arn a E E T AA E 16 TFABEE eee a E E a 19 INTRODUCTION parerii a a E E 20 Z QUICK TARF aen ition qceonaseniaisidermenaiiaamntoncmencmoddiest 22 Ziel PUING eaire ea a E iA EE E A a AAEE T
143. ng or reducing the HV of the PMT detector or gain on the output of the LAAPD the detector until the optinum photometric DC voltage is achieved regardless of incident light intensity This best matches the digitisation stage and results in the best quality CD resolution In regions of the spectrum where there is a high photon flux high light level the required HV will be relatively low whereas in regions of the spectrum where there is a low photon flux for example in the far UV the HV PMT or gain and associated emulated HV LAAPD will be increased To protect the PMT detector the maximum HV is 1000 volts which will be reached at the very lowest light levels Note that at the very lowest light levels the emulated HV on the LAAPD will also reach 1000 volts which is a gain of 4096 over the internal detector gain and the maximum permitted lt should be noted that if the HV or emulated HV has reached its maximum level and the incident light continues to fall the CD signal can no longer be further optimised However even with reduced signal levels a good CD signal can still be measured A very high HV or emulated HV is not necessarily an indication that there is no CD signal 6 4 3 The adaptive sampling algorithm Adaptive sampling aims to optimise the overall scan time by spending more time collecting data where it is most needed which means in regions of low light intensity and to scan more quickly where light is plentiful This function has bee
144. nged as the scan progressed being high where the sample absorbance is high and low where the absorbance is low Clicking the Advanced button will cause a password prompt to appear and users should not proceed except under instruction from an APL engineer 4 13 5 Device icons requiring accessories The device icons below require accessories requires a CS TF fluorescence detector see the Fluorescence Accessories User Manual Fluorescence requires the CS SEM scanning emission monochromator see the Fluorescence Accessories User Manual requires the CS TT dual syringe titration unit see the CS TT User Manual E E a 5 requires an CS SF stopped flow accessory see the CS SF User Manual a Een ee i requires the CS PCS single cell Peltier temperature controller see the CS PCM User Manual Fo g 3 a pm C requires the CS PCM 4 cell autochanger see the CS PCM User Manual E 5 Document 4207Q108C03 01 Page 78 of 133 Applied hotophysix Ultrasensitive Spectroscopy for the Life Sciences Bieter Peter EE 3 259 Chirascan Series User Manual 4 14 The Preferences dialog box Clicking on the Preferences icon or selecting Preferences from the Configuration drop down menu will display the Preferences dialog box 4 14 1 The Viewer tab Preferences Viewer CD Absorbance Fluorescence Data Acquisition File Names Startup Preferences Pro Data Viewer Preferences Z Dont
145. nit ESHU panel Figure 4 18 shows the temperature of one of up to four temperature probes T1 to T4 selected from the drop down menu in the above figure probe T1 is registering 23 5 C The temperature measured by the chosen probe is one of the properties recorded in the Datastore Click the Setup button to reveal the Multi cell Sample Setup dialog box Figure 4 19 Document 4207Q108C03 01 Page 69 of 133 AppliedPhotoph Ultrasensitive Spectroscopy for the Life Sciences Se ea eee AT Chirascan Series User Manual Multi cell Sample Setup F Enable Multi cell AutoChanger Check all used cell positions sample Description Sample 1 Pathlength 10 and Cell Tupe Position 2 Sample Description Pathlength and Cell Type Position 3 Sample Description Pathlength and Cell Type Position 4 Sample Description Pathlength and cell Type Figure 4 19 the Sample and Cell description dialog box Do not tick the Enable Multi cell AutoChanger box unless the PCM 9 is fitted Use the edit boxes to enter a description of your sample and cell The information is stored in the Datastore and can be viewed in Pro Data Viewer Section 5 1 3 The Titration button is inactive unless the CS TT titration accessory id fitted and the Stirrer button is inactive unless the CS PCS or CS PCM are fitted For more information see the User Manuals for those devices Document 4207Q108C03 01 Page 70 of 133 Applied Ultrasensitive Spectro
146. not occur Circular Dichroism mdeg ha 180 190 200 210 220 230 240 250 260 VVavelength nm Figure 5 41 multiple lysozyme spectra Document 4207Q108C03 01 Page 110 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences E rs Pedr AAA E EA Chirascan Series User Manual There are approximately 400 spectra of lysozyme in Figure 5 41 The experiment was to monitor the photolysis of the protein over time Each spectrum was measured in about 90 seconds The secondary structure changes with time and it is therefore not appropriate to average the spectra but we are interested only in the random noise on the spectra Noise can be separated from a spectrum by smoothing the spectrum and keeping the residual raw data smoothed data Taking the average of the residuals is a valid operation and shows that the random noise is dramatically reduced by the process of averaging theory tells us that a 20 fold improvement in signal to noise ratio S N would be expected from averaging 400 observations 0 5 0 5 0 5 0 5 Circular Dichroism mdeg Circular Dichroism mdeg ee 180 190 200 210 220 230 240 250 260 i 180 190 200 210 220 230 240 250 260 VVavelength nm VVavelength nm Figure 5 42 smoothing residuals left and their average right Repeating the above process with a single background subtracted from each spectrum prior to generating the residuals yields a quite different picture The average re
147. nput When converting from mdeg or delta A to units that are normalized in some way further input is required To convert to molar delta A or molar ellipticity the calculation normalizes for and therefore requires knowledge of the molarity of the solution and the pathlength of the cell cm Figure 5 16 Document 4207Q108C03 01 Page 96 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Fee he A AF Chirascan Series User Manual Convert CD Units Convert From mdeg Convert To mean residue molar ellipticity Concentration mgm Path Length cml Residue Mw tenet Figure 5 16 converting to a normalized unit For proteins it is usual to normalize for the number of amino acid residues as well as concentration and pathlength When Mean residue molar delta A or Mean residue molar ellipticity are selected the input required is the concentration of the protein mg ml the pathlength of the cell used cm and the mean residue molecular weight The default input for the Residue MW is 113 a commonly accepted value for the mean molecular weight of an amino acid residue This default value can be changed if required The units are presented in this way to make it possible for proteins that are not yet fully characterized to be normalized for comparative purposes 5 3 4 Saving data The manipulated data are not saved automatically To save the result go to Save Current Plot on the File menu Section 5 7
148. nrernnnnne 45 Table 2 10 operating conditions for the spectrum in Figure 2 29 nnannnannnnnnnnnnnnnnnnnnnnornnnnnennnrronnrnnnrrnnrrrenerenenne 46 Table 2 11 operating conditions for the spectrum in Figure 2 30 ccc ceecccceececeeeeeeeeeeceeeeeceueeeseeesseeeessaeeesaeeeees 47 Table 3 1 module labels for the standard CONFIQUIATION cccceeecccceeeececeeeeceeceeeeeeceeeeeesseseeeeseeeceeseaeeeessaeeeessnaeees 50 Table 4 1 conditions for CD calibration ccccccssccccessseeceasseecceseeecsaseeecsauseecsauseeessaececsauseecsaseeesegeressageeecsageees 82 Table 5 1 operating conditions for the spectra shown in Figure 5 49 eeeccccseeeeecseeeeeeseeeeeesaeeeeesseeeeesaaeeeeseaes 115 Document 4207Q108C03 01 Page 19 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences sch faire reset eran A 5 apes Chirascan Series User Manual 1 INTRODUCTION Figure 1 1 the Chirascan plus spectrophotometer Circular dichroism CD is the difference in the absorbance of left handed circularly polarised light L CPL and right handed circularly polarised light R CPL and occurs when a molecule contains one or more chiral chromophores light absorbing groups i e CD AA A A A icer A A rceL where A is the wavelength Circular dichroism CD spectroscopy is a spectroscopic technique where the CD of molecules is measured over a range of wavelengths CD spectroscopy is used extensively
149. nt 4207Q108C03 01 Page 48 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences FE SR Sar eae Re Ct pe Chirascan Series User Manual 3 HARDWARE This Chapter describes the Chirascan hardware covering the general layout of the spectrometer the electrical and purge gas connections and the detectors and light sources available and their removal and replacement 3 1 The spectrometer layout Monochromator and electronics Lamp housing cover Sample Handling Unit ESHU 0 rbot physics A lt 2 Z res Ti EE Figure 3 1 the spectrometer layout The Chirascan series spectrometers comprise three main sections as described in Chapter 1 of this User Manual and identified in Figure 3 1 the lamp housing on the left the monochromator and electronics in the centre and the Equilibrium Sample Handling Unit ESHU on the right The lamp housing and monochromator electronics sections have removable covers fitted the ESHU is a self contained unit that can easily be removed for transportation or to install the CS SF stopped flow unit 3 2 Electrical connections WARNING The Chirascan series spectrometers are powered by the mains electricity supply which can produce a shock leading to serious injury or death Do not connect or disconnect the instrument from the mains supply unless the supply is powered off at Source Exercise care during operation and do not operate units with their covers re
150. o its greater stability and higher output at specific wavelengths The low wavelength radiation produced by this lamp is responsible for the conversion of oxygen to ozone and this lamp should only be used with nitrogen purging For more information on the use of this lamp see the CS SF User Manual 3 4 4 Ozone free xenon lamp A non ozone producing xenon lamp is also available The low wavelength radiation responsible for the conversion of oxygen to ozone is filtered from the lamp output removing the need for nitrogen purging but limiting the low wavelength end of the range of the lamp to about 230 nm Document 4207Q108C03 01 Page 52 of 133 Applied hotophysix Ultrasensitive Spectroscopy for the Life Sciences OPP eee eee eee eee eee eee Chirascan Series User Manual 3 4 5 Lamp removal and replacement The xenon arc lamp operates at mains voltage Power off the lamp before removal Xenon arc lamps have a high internal pressure and can burst particularly when hot causing injury to the user Handle carefully do not submit the lamp to shock and wear eye and face protection Do not touch the glass bulb of the lamp with bare hands The lamp is at high temperature during operation After powering off allow at least 30 minutes to cool before removing the lamp Leads End cover Retaining screws Figure 3 5 lamp housing fittings To change a lamp follow the steps below referring to Figure 3 5 hk 11 12 Have a
151. o the instrument via a manifold containing three gas flow controllers Figure 2 1 The gas pressure regulator of the master supply should be set in the range 4 to 6 bar 60 to 90 p s i Figure 2 1 the purge gas manifold gas flow controllers The three gas flow controllers rotameters control nitrogen flow to the lamp housing monochromator and sample housing respectively from left to right Document 4207Q108C03 01 Page 22 of 133 AppliedPhotophysix Ultrasensitive Spectroscopy for the Life Sciences Spc AE A ER 5 ep Chirascan Series User Manual At first start up set the gas flow to 4 L min 10 L min and 4 L min for the lamp housing monochromator and sample chamber respectively and let the purge continue for at least 30 minutes before attempting to ignite the lamp Section 2 2 After this initial high flow purge the controllers can be set at their normal operating flow rates of 1 L min 3 L min and 1 L min The flow rates are summarized in Table 2 1 N flow rates N flow rates start up L min normal L min Lamp housing Sample chamber Total Table 2 1 No purge flow rates Note that the maximum possible flow rate will be determined by the source pressure If the pressure is significantly below the maximum 6 bar then it might not be possible to achieve the listed flow rates If that is the case set the flow rates to the highest possible directing the majority of flow through the monochromator The lamp housing a
152. obtain each data point Selecting the Logarithmic option results in the logarithmic distribution of data points over the selected acquisition time This feature may be useful when the kinetic change being monitored has a fast initial phase but a slower second phase As an alternative a Split Timebase may be used in which data points are distributed between two linear intervals 4 6 The Sampling panel Sampling Time per paoint a 0 5000 Approximate scan time 73 seconds Current Time per point s 0 5000 Enable Adaptive Sampling Figure 4 12 the Sampling panel The Sampling panel Figure 4 12 is visible when operating in Spectrum mode and is used to control the sampling time for each point in a scan Use the Time per point s edit box to specify the time in seconds to be spent collecting data at each point an approximate scan time is calculated from the product of the time per point and the number of points in the scan specified in the Monochromator panel Section 4 4 If the Enable Adaptive Sampling check box is ticked the time per point is varied inversely as a function of the light reaching the detector Where the signal is weaker more time is spent collecting data than where it is Document 4207Q108C03 01 Page 66 of 133 Applied hoto Ultrasensitive Spectroscopy for the Life Sciences E eh eee SP Chirascan Series User Manual stronger The algorithm has been developed such that it will usually take less time to measure a s
153. open blank graph window when starting Pro Data Viewer V Auto scale plots during data acquisitions Figure 4 30 the Viewer tab The dialog box opens at the Viewer tab Figure 4 30 where the appearance of Pro Data Viewer when launched can be controlled with or without the blank display and whether or not the display automatically rescales when a data point is measured that is outside the limits of the currently displayed y axis 4 14 2 The CD tab i Preferences Viewer CD Absorbance Fluorescence Data Acquisition File Names Startup Preferences Default Mode Auxiliary Measurements J delta A HV millidegrees AutoAbsorbance V Enable AutoAbsorbance by default Figure 4 31 the CD tab On the CD tab Figure 4 31 preferences for CD measurements can be set In the Default Mode panel select either delta A delta absorbance or millidegrees as the units for expressing circular dichroism in the Auxiliary Measurements panel HT is greyed out and permanently selected i e the detector HV Chirascan or emulated HV Chirascan plus will always be recorded in CD mode and in the AutoAbsorbance panel Enable AutoAbsorbance by default can be selected which is recommended Document 4207Q108C03 01 Page 79 of 133 Applied hotophysi Ultrasensitive Spectroscopy for the Life Sciences Betas AE rae tats 3 9 Chirascan Series User Manual 4 14 3 The Absorbance tab r Preferences Viewer CD__
154. oscopy for the Life Sciences Pre rps a eon 33 Chirascan Series User Manual CD Signal Type Please indicate type of data units Be carefull Wrong assignment will result in incorrect data renormalization CD Signal Type Essential Parameter C Delta Epsilon Molec mass Da 1 4800 0 C Molar Ellipticit Concentr mg ml 0 200 olar Ellipticity Mo amino aa Mill Degrees A ms mo Figure 5 52 the CD Signal Type window With Millidegrees selected the Essential Parameter panel appears Figure 5 52 It is important that the information requested is supplied For lysozyme measured under the conditions given above the fields are correctly filled in Click OK and then Deconvolute F Deconvolution File Edit Help C D a W w D m 210 220 230 240 Wavelength nm Figure 5 53 the Deconvolution window The results will appear in the Deconvolution window Figure 5 53 Comparison of the example and original data will show that the results are broadly similar There are small differences in the spectra and it is not therefore surprising that there are small differences in the calculated contributions of the various secondary structure components Document 4207Q108C03 01 Page 117 of 133 AppliedPhotoph Ultrasensitive Spectroscopy for the Life Sciences Chirascan Series User Manual 5 10 3 Dealing with uncharacterised proteins Not all proteins are well characterised for example the molecular weigh
155. oved and an alternative sample handling system configured for example to install the CS SF stopped flow accessory A lid open detector is fitted as standard which will pause an experiment in progress and reduce the photomultiplier high voltage to prevent damage if the lid is raised 6 1 3 The LAAPD detector Chirascan plus only The feature that distinguishes the Chirascan plus from the standard Chirascan is the solid state large area avalanche photodiode LAAPD CD detector that replaces the photomultiplier tube PMT detector on the standard instrument Avalanche photodiodes are effectively the solid state equivalents of photomultiplier tubes Using a high reverse bias voltage the detectors exhibit a high internal gain in the same way that high voltages to photomultipler anodes boost the internal gain of photomultipliers Large area avalanche photodiodes are a new generation of APDs with features that make them high performance replacements for photomultipliers for many applications These features include a large light collection area similar in size to photomultiplier photocathodes higher quantum efficiency than photomultipliers extended wavelength range sensitivity and rugged construction and high internal gain The LAAPD detector on the Chirascan plus is a 1 cm diameter round detector windowless silicon APD The detector shows a higher quantum efficiency i e a higher number of displaced electrons for each incoming photon throu
156. own in Figure 2 23 The operating conditions are given in Table 2 4 z f C Program Files x86 Applied Photophysics Chirascan Examples Alcohol dehydrogenase dsa Circular Dichroism D ecje A File View Trace Math Scale Window Help GS Ei Cod CD OS 9 Circular Dichroism mdeg 190 200 210 220 230 240 VVavelength nm Figure 2 23 CD spectrum of alcohol dehydrogenase Table 2 4 operating conditions for the spectrum in Figure 2 23 Document 4207Q108C03 01 Page 40 of 133 AppliedPhotophysics Ultrasensitive Spectroscopy for the Life Sciences 2 9 2 Bovine serum albumin Chirascan Series User Manual The CD spectrum of bovine serum albumin plotted as CD mdeg against wavelength nm is shown in Figure 2 24 The operating conditions are given in Table 2 5 E C Program Files x86 Applied Photophysics Chirascan Examples Bovine serum albumin dsa Circular Dichroism 5 8 A File View Trace Math Scale Window Help ol x EREL 2 S e 4 Circular Dichroism mdeg 180 190 200 210 220 230 240 250 260 VVavelength nm Ready NUM OO p Figure 2 24 CD spectrum of bovine serum albumin Scan range 3 seconds Table 2 5 operating conditions for the spectrum in Figure 2 24 Document 4207Q108C03 01 Page 41 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences 2 9 3 Cytochrome C Chirascan Series User Manual Th
157. ozyne Spect About Pr Data Viewer Figure 5 33 the Help menu Document 4207Q108C03 01 Page 105 of 133 AppliedPhotoph Ultrasensitive Spectroscopy for the Life Sciences 5 8 Graphical Display drop down menus 5 8 1 The File menu The File menu Figure 5 34 gives access to the usual Windows file handling options C Program Files x86 Applied Photophysics Chirasce E View Trace Math Scale Window Hel G Saved Save Current Plot Shift S Save Selected Traces Ctrl Shitt S Copy to Clipboard Ctrl C Print Ctrl P Close Figure 5 34 the File menu 5 8 2 The View menu CAFrogram Files x86 Applied Photophysic E File Trace Math Scale Winc Gil E Aux Dimension Ctrl v Legend L Remarks History Figure 5 35 the View menu On the View menu Figure 5 28 Chirascan Series User Manual e Aux Dimension Ctrl is a toggle to show or hide the third dimension slider bar It is of use when dealing with multi dimension Datastores e Legend is a toggle to hide or show the trace names that appear by default at the side of a plot The function can be more easily executed using the keyboard shortcut L or by right clicking anywhere in the plot border and clicking on the Hide legend or Show legend pop up that appears e Remarks calls up an edit box in which you can add comments to supplement information that is recorded automatically or entered through the Setup dialogue box in the SCP e
158. pectrum than at constant time per point Clicking on the Adv button calls up the Sampling Advanced dialog box Figure 4 13 Sampling Advanced Sample period us 25 0 Y Default number of samples 20000 Disable oversampling Adaptive sampling limits Minimum number of 100 samples Masimum number of 4000000 samples PRT low H warming limit 230 Save Figure 4 13 the Sampling Advanced dialog box The Sample period us can be set at any value between 10 0 us and 1000 0 us in the edit box followed by clicking on the Apply buiton The Default number of samples can be set anywhere between the minimum and maximum limits see Sampling limits below The product of the sample period and the number of samples yields the current time per point The default number of samples is superseded by changing the Time per point in the Sampling panel as described above Selecting Disable oversampling will do precisely what it says and is used for diagnostic purposes only If it is selected a warning is given Figure 4 14 for all non diagnostic use oversampling must be used Chirascan _ Warning Disabling the oversampling can seriously degrade data eK acquisition performance Figure 4 14 the Disable oversampling warning message Document 4207Q108C03 01 Page 67 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PES RET ESP Chirascan Series User Manual
159. pplied PC However APL cannot be held responsible for failure of the software or hardware through of misuse or neglect by the customer If you have any queries relating to the Chirascan software or the HP Compaq PC please contact APL Technical Support at the address given on the front of this User Manual Servicing servicing of the Chirascan and its accessories should only be undertaken by qualified personnel If you are in any doubt at all please contact the Applied Photophysics Technical Support Department at the address given on the front of this User Manual Document 4207Q108C03 01 Page 10 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual GLOSSARY The following abbreviations are used in the descriptive text in this User Manual AC Alternating current APL Applied Photophysics Ltd AU Absorbance units CD Circular dichroism CPL Circularly polarized light DC Direct current FWHH Full width at half height HT High tension the same as high voltage HV High voltage L min Litres per minute M Molar i e mol dm SBW Spectral bandwidth SF Stopped flow SS Step size UV Ultraviolet The following abbreviations relate to components of the Chirascan spectrometer APD Avalanche photodiode ESHU Equilibrium sample handling unit FDCD Fluorescence detected circular dichroism KSHU Kinetic sample handling unit LAAPD Large area avalanche photodiode LED Light emitti
160. protected to prevent unauthorised interference as calibration data and motor drive parameters can be modified They are provided to allow complete instrument control on site by APL personnel and to allow tests to be run by customers under APL supervision 6 3 Simultaneous calculation of absorbance and CD 6 3 1 Introduction The simultaneous measurement of absorbance during a CD measurement is a useful and important facility on the Chirascan series spectrometers The absorbance spectrum provides an indication of the transmission of the sample and any solvents present over the wavelength range under investigation This is helpful in judging whether a CD measurement is valid since a lack of light throughput will directly result in an erroneous CD due to the attenuation of AC and DC signals to meaningless levels This will usually manifest itself as a marked rise in the noise on the CD spectrum but this alone may not at first glance indicate the degree of deterioration in the validity of the measurement This is particularly problematic in the far UV region of the spectrum where many buffers are opaque but CD information is of particular interest e g for the secondary structure analysis of proteins The absorbance of many common buffers and salts rises very sharply below 200 nm and this wall which is clearly evident in the absorbance spectrum indicates to a first approximation the wavelength limit of detection The absorbance spectrum
161. r compounds a different wavelength range would be used Place a cell containing distilled water in the sample compartment Select Circular Dichroism from the dropdown menu in the Signal panel and click Background Before removing the blank select Absorbance from the drop down menu in the Signal panel and click Background again Now replace the water with the sample Without changing anything click Acquire to measure the true absorbance spectrum Now select Circular Dichroism again Click Show in the Background panel and Load to reinstate the CD background measured previously With the CD background loaded click Acquire to measure the CD spectrum and derived absorbance of the sample To see the derived absorbance spectrum right click on the y axis legend and select Absorbance Use the cursor to determine the absorbance maximum of both the derived and the true absorbance and from these determine and apply the correction factor as described above Remember that the derived absorbance is only an approximation Its function is to provide you with a reasonably accurate indication of whether or not you are within acceptable absorbance limits when measuring a CD spectrum The scaling factor will vary slightly with wavelength ensure the scaling factor is calculated using a wavelength as close as possible to where most of your measurements will be carried out The factor will change slightly as the detector ages Occasional checking is advisable The fr
162. r of samples and therefore the default time per point used during normal and adaptive sampling Minimum and maximum sample sizes can also be modified to limit the sampling extremes during adaptive sampling Document 4207Q108C03 01 Page 131 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE ee eee Chirascan Series User Manual In conclusion the adaptive sampling option will not always offer significant benefits but with experimentation will result in scan time improvements for certain samples and scan wavelength ranges 6 5 Data filtering 6 5 1 Introduction Signals derived from photomultipliers inevitably have photon shot noise superimposed which is due to the random nature of photon detection events at the photocathode This is of high frequency and Gaussian in distribution and is usually the most significant type of random noise contributing to the overall noise in a CD measurement Other sources of noise include electronic noise and noise which arises from the PEM modulation frequency of 50 kHz There are three distinct forms of signal filtering which are implemented or available on the spectrometer e Signal bandwidth limiting and noise rejection in the analog electronics e Post acquisition signal averaging for shot noise reduction where signals are weak e Cosmetic smoothing of experimental results for presentation The first of these is an essential and optimized part of the processing of the raw analog signa
163. r thumbscrews Figure 3 7 To remove the ESHU disconnect the nitrogen purge gas connection Section 3 3 release the thumbscrews and slide the ESHU to the right away from the monochromator Do not alter the height settings on the ESHU feet To replace the ESHU perform the removal operation in reverse The ESHU and monochromator should align precisely if they do not make the necessary height adjustment using the three adjustable feet beneath the ESHU not those on the monochromator 3 6 Detectors 3 6 1 CD detector options The Chirascan uses a photomultiplier tube PMT for CD detection The Standard PMT has an operating range from 165 nm to about 900 nm The longer wavelength limit in the near IR can be extended with the IR3 Extended NIR PMT accessory which is suitable for use over the range lt 250 to 1250 nm The Chirascan plus uses a large area avalanche photodiode LAAPD detector with an operating range from 165 nm to about 1360 nm 3 6 2 Removal and replacement WARNING The photomultiplier tube PMT detector used with the Chirascan and the large area avalanche photodiode LAAPD detector supplied with the Chirascan plus operate at high voltages and can produce a shock leading to serious injury or death Do not connect or disconnect the detector from the spectrometer unless the spectrometer is powered off To remove a detector ensure that the spectrometer is powered off and disconnect the cable Loosen the knurled locking ri
164. r will be established automatically When launched in this way Pro Data Viewer will be online and becomes a live display for data as they are collected although it can still be used for display and manipulation purposes lf Pro Data Viewer is started independently of the SCP using the desktop icon and you subsequently wish to establish a link between the programs this can be done in the Preferences window Section 5 7 4 5 1 2 File structure the Datastore Experimental data are written to a results file called a Datastore which is a proprietary format designed for handling multi dimension data Its content will depend upon the type of experiment that is carried out A knowledge of the structure of Datastores will be helpful in understanding some of the functionality of Pro Data Viewer A Datastore consists of dimensions and properties The dimensions are the controlled independent experimental variables such as wavelength or time the properties are the measured dependent variables such as CD or absorbance temperature may be either a dimension or a property depending on the type of experiment When data are plotted using Pro Data Viewer dimensions are always plotted on the x axis and the properties on the y axis A Datastore can therefore have one or more dimensions depending on the number of controlled variables For example a simple experiment in which CD is measured as a function of wavelength would generate a one dimension Data
165. raw data cccccccccseeeeceeceeeeeeeeeeeeeeeeeeeseeeseeeeseeeaeeeeeeseaeeeeessaaeeeeesssaaeeeees 30 FIGUKG 2 13 We VVIMGOWS dlalog BOX cases ctdacraetacncneac sisenaccwsace a D eunedceaaaeetioie dua ETE EN 31 Figure 2 14 Raw data with dialog box called up cccecccccccssseeeeeeceeeeeeeeeeeeeeeeeeeeeeceeeseeeeeeeeeesaeeeeeessaaeeeeeessaaeeeees 32 Figure 2 15 an appropriate smooth left and an inappropriate one right cee cceeecseeeeeeeeeeeeeeeeeeeeaaeeeeeeeaaaeeeess 33 Figure 2 16 Trace manipulation dialog box after spectrum manipulation cece cceeeceeeeeeeeeeeeeeeeeeeaeeeeeeesaaeeees 34 Figure 2 17 smoothed and background corrected spectrum of lysozyme red and smoothing residuals cyan 34 Figure 2 18 the Datastore History citcessantusonncctaisinennstbantcadsiusteasiienressdibentasiubousnensiesiantalicunscadessdaedeanieadsbesncteutuntaatiunies 35 Figure 2 19 File manipulation pop up MENU cccceeccccseseeecceescecceseeeceuseeecseececseuseeessaeeeessageeessageeeesesseesssegeeenes 35 Figure 2 20 options for exporting data cccccccccsescccceeeeeecneeeeeeseeeeeeeeseeesseeeeeeseeeeeseaeeeesseeeeesseueeessaeeeeeseeeeeesseneesens 36 Figure 2 21 the APLDataConverter target ccccccccseeeeceeeceeeeeeeeeeaeeeeeeeeeeesceeeeseesseeeeseeesseeeessaeaeeeeessaaeeeesessaaeeeees 36 Figure 2 22 the APLDataConverter Option dialog DOX cccceceeeeesecceceeeeeeeseeeceeeeeesaaeeeeeeeeeees
166. re as an emulated HV providing consistency and transparency in the user interface between the Chirascan and Chirascan plus The relationship between the Chirascan plus amplifier stage gain and the emulated HV is shown in Figure 6 4 10000 1000 100 10 Amplifier stage gain t200 0 01 Emulated HV Figure 6 4 LAAPD amplifier gain and emulated HV value Document 4207Q108C03 01 Page 122 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FES eee Os Chirascan Series User Manual Dark current in the LAAPD detector is autocorrected within the electronics and software At the start of data acquisition when the autoshutter is closed a dark current measurement is made and a correction factor set This backoff is automatically adjusted by monitoring changes in the temperature of the photodiode and using a temperature correction factor to adjust the dark current correction factor High levels of light hitting the detector will produce a high signal current Sustained high levels of light produce a large current that can overload the photodiode element To prevent this the internal gain of the detector can be reduced by lowering the reverse bias voltage This has been set to a level to allow safe operation of the instrument at a 2 nm spectral bandwidth by the APL production department As a precaution the electronics can detect an excess light condition warning the user and turning o
167. re detailed information on these operations is given in Chapters 4 and 5 of this User Manual 2 1 Purging A CAUTION The interaction between ultraviolet light and oxygen leads to the formation of ozone a very reactive gas that is damaging to health and may cause deterioration of the optical components of the instrument If an ozone producing lamp is used it essential that the spectrometer is thoroughly purged with nitrogen before the lamp is ignited Oxygen in the form of Os absorbs light in the far UV region of the spectrum and its presence will reduce the wavelength range of the spectrometer More importantly the interaction between oxygen and UV light leads to the formation of ozone O3 which absorbs in the UV at higher wavelengths than oxygen further reducing range of the instrument and is also damaging to health and to some of the components of the instrument If an ozone producing lamp is used it is essential that the instrument is thoroughly purged with nitrogen before the lamp is ignited and remains purged while the lamp is on lt is also essential that the nitrogen used to purge the instrument is clean A combined molecular sieve 13x and activated charcoal filter is supplied with the instrument which removes from the nitrogen stream all common impurities that could otherwise deposit on the internal optical surfaces This filter should not be by passed or removed After passing through the filter the nitrogen is distributed t
168. ribed below System Reset will reset any user adjustable parameters accessible through the Devices panel to their factory defaults It is unlikely that you would need to do this Calibrate Zero Offsets will adjust the electronic offset to take into account any small drift that may have occurred over time It is unlikely that you will need to this on a regular basis The offsets should not be changed until the instrument electronics are at their normal working temperature it is good practice to leave the electronics powered on at all times nor should they be changed part way through a series of experiments since recalibrating the offsets may shift the CD background slightly Service Password opens a dialog box that permits you to change the password that gives access to the advanced sections of the Device window If you change it do not forget it because APL engineers will not be able to rediscover it for you Document 4207Q108C03 01 Page 74 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences 4 13 2 Mono1 P lt the Mono1 icon displays the Monochromator window Figure 4 27 Mona Monochromator Mono 1 Wavelength rm p Bandwidth nm Calibration File Status Mono lD EUMonoz Reached target Curent action Ready Yeg Temperature C 27 1 a C Program Files x86 4oplied Photophysics Chirascan Configi Devices M onoCal cont Calibration temperature 23 00 C Reload calibration
169. rivileges the Calibration button in the CD signal Background panel will be active Click on it to open the CD calibration dialog box Figure 4 37 and make a note of the current Desired CD value mdeg and Calibration value then if necessary these values can be reinstated and activated by clicking Apply and then OK CD Calibration gt Desired CD value mdeg 190 4 Calibration Value 1152 37 Lok Cancel Apply Help Figure 4 37 the CD calibration dialog box Type the CD value you have just calculated for your CSA solution into the Desired CD value mdeg box and click Measure The calibration procedure will take a measurement of the CD at 290 5 nm and adjust the Calibration value automatically If you are happy with the outcome click Apply and then OK Check that the calibration is correct by measuring the CD spectrum of your CSA solution over the range 320 nm to 260 nm using 0 5 nm steps and use the cursor in Pro Data Viewer to establish that the observed and calculated values at 290 5 nm are the same 4 15 2 Recalibrating the derived absorbance Chirascan only There is no derived absorbance on the Chirascan plus the absorbance measured in CD mode is derived directly from the detector gain and is identical to that measured in direct absorbance mode or on a dedicated UV visible spectrometer On the Chirascan the derived absorbance calculated during a CD measurement is an approximation which will be reasonably
170. s menu Figure 2 13 Windows Select Property CircularDichrom HT Absorbance Temperature Voltage Court Cancel Figure 2 13 the Windows dialog box 2 5 2 Comparing spectra If you wish to compare spectra drag and drop the files of interest onto the graphical display window from the Datastore Explorer More than one spectrum at a time can be dragged using the usual combinations of Ctrl Shift and mouse buttons The spectra will be displayed in a single window Note that the data sets must be compatible you cannot overlay a trace collected as a function of wavelength with one that was collected as a function of some other variable such as temperature 2 5 3 Manipulating spectra In the example below there are six traces 3 repeat backgrounds and 3 repeat lysozyme spectra The data used in this example can be found in The Examples Lysozyme spectra folder in C Program files Applied Photophysics Chirascan We will average the backgrounds average the spectra subtract the average background from the average spectrum smooth the resulting background corrected lysozyme spectrum and remove the original data The end result will show the lysozyme spectrum and its residual calculated minus observed spectrum With the data loaded into the display as previously described and the display window active click on 2 to call up the Trace manipulation dialog box Figure 2 14 Document 4207Q108C03 01 Page 31 of 133 Applied
171. s the basis of the Chirascan s adaptive sampling mechanism Implementation of the approach required making measurements of the noise variance of a typical detector as a function of HV Some sample results are shown in Figure 6 5 in which noise variance is plotted against HV at constant photometric DC target A clear non linear increase in signal variance with HV can be seen between 200 and 1000 volts the usual operating range of the detector Noise variance 0 100 200 300 400 500 600 700 500 900 1000 HV volts Figure 6 5 noise variance against HV at constant DC target The data were then used to construct a plot of required relative average sample size required to maintain a constant noise level versus HV Fitting an empirical formula to this data yields the continuous curve shown in Figure 6 6 enabling a relative sample size to be calculated for any HV Document 4207Q108C03 01 Page 130 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences ee ne SE EAT Chirascan Series User Manual Rel sample size 0 100 200 300 400 500 600 700 a00 900 1000 1100 HV volts Figure 6 6 relative sample size as a function of HV To make use of this function we first define a default sample size and reference HV which act as the starting point for the adaptive algorithm These values determine a default time per point and noise level which is broadly acceptable for typical spectral measurements into the far UV
172. saeaeeeeeeeessssaaaseeeeees 37 Figure 2 23 CD spectrum of alcohol dehydrogenase cscecccccccaseeeeeeeeaeeeeeeeeeeeeeceeeseaeeeeeeesseeeeeeeessaaeeeeeessaeeeees 40 Figure 2 24 CD spectrum Of bovine serum albumin cccccseeeececcceeseeeeecceaeeeeeeceaassceeesseasceeessaaeceeessaaeeeeeessaaeeees 41 Figure 2 25 CD spectrum Of cytochrome C ou cc ceecccccseeeeecaeeeeeeneeeeeecaeeeeeeaeeeeeeeaeceeseaeeeesseueeessaeeeessaaeeeeseaeeeesseneenens 42 Figure 2 20 GU spectum Of IVSOZYING sinistre EE ET EEE deitianetod 43 Figure 2 27 CD spectrum of vitamin B12 eeeccccccseseeceecceeeeceeccaesseeeeeeseaseeeeseseaseeeesseaaeeeeessaaeeeeeessaaeeeeessaaaeeeees 44 Figure 2 28 CD spectrum of tris ethylene diamine cobalt chloride cecccccccsseeeeeeeceeeeeceeeeeeeeeceeesaaaeseeeessaaeeees 45 Figure 2 29 CD spectrum of camphor sulphonic ACIC cceeeceecceeeeeeeeeeaeeeeeeeceeeeseeeessaaseeeeeesaeeeceeessaaeeeeeessaaeeeees 46 Figure 2 30 CD spectrum of R 3 methylcyclopentanone ccccceeececcseseeeceeeeeeeeeeseeeeeaeeeesseaeeeesaseeeesaeeeesseeeseas 47 Figure 3 1 the spectrometer layout sirevicatutnsncandienneanstantcadsiusicassianteneniensavialundsnatiesnentsiwntinduidevdaeditusicntdiertentavantsaauleste 49 Figure 3 2 mains electricity CONNECTIONS cccccsccccceeseecceeseeecceuseecceseeeseuseeecsaseecsauseeessseeessaaeeessageesesseseesssageeenes 50 Figure 3 3 the purge gas inlet connection on the rear of t
173. saueeeesssaueeeeesseaueeeessseageeeeessageesesssaages 72 BAZ We TAS FSi SIME E E E nena emcee sie eny seca AOE A A O A 72 A Te VICE E EE EE E E E E E E E AEEA EN 72 ARo The Connieuri Onine aien E E EN AE 73 AATE AED MIE a E E E N EE E A O TT 73 4 13 The Device WINKOW cccceeececcseseecceesseccceusceeceaeeecsauececseuseeessugeeeesaseeeseuseeessageeessaeeeesegseeessageeessagseesseass 74 Os VGN EEE E E E EE A E A E sear ous aco seer A T A E EET 74 ALA IW VON Oh cetsens Sate says ceed ain n E N N E E EE E S 75 A TNS ae N O ea E E E E 76 4 13 4 The circular dichroism DC Channell ccccsssccccsssecccenecccsenscccauscccsaecesseueceseaeecesauesessenecessueseeseueeessanseess 77 4 13 5 Device icons reguiring accessories sccsseccseccesccessccssccesccessccsccesecssecseccesecsseesaesceseceaecsaescesscsaeesaesensnes 78 4 14 The Preferences dialog DOX ccccccccccccssssecececeeeecececaeeseceeecseeeeeeeseaaeeeeesaaueeeeessuaeseeeessuaaeeeeessaageeeesssaaess 79 a e T CWC e E EAEE E I E E E T 79 aN SDI o gt e AE E E eee ee A ee E E eee ee eee 79 EE he WV FAS ON AC ea a A E A E T tes cia E E E E E E E ae Rea 80 A NAA TG FIUGLESCOMCE Ta egeria 80 Document 4207Q108C03 01 Page 13 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences pesnneeskeatenerserenens Chirascan Series User Manual 14 5 Te Data AC OUISILION TAD sass scnccuwatonoiressenercocsanannostanscaamesiesaunalf E EE EE EAA EO RE ioneneatereea
174. scopy for the Life Sciences BEE are re eee Ree On Chirascan Series User Manual 4 11 Toolbar icons Figure 4 20 the SCP Toolbar The SCP toolbar is shown in Figure 4 20 Positioning the cursor over an icon will call up a brief description a fuller description is also provided in the status bar at the foot of the SCP The action of clicking on each icon is described below Opens a new document Opens a previously collected data file This will open a window showing the contents of the currently active directory or the examples folder if you have just launched Pro Data Opening a data file from this window will configure the interface with the experimental parameters contained in that file Saves the active document If there is new data that needs saving this icon will open a Save As window showing the currently active directory If there is no new data an information window saying so will appear Opens the File Names dialog box in which you can specify seed names for spectra and backgrounds In a series of experiments the file names will share the chosen seed name and be differentiated by an automatically incrementing numeric component e g Protein0001 Protein0002 A Opens the Remarks and Comments window in which you can record information about your experiments The remarks are stored in the Datastore and can be viewed in Pro Data viewer Opens the Preferences dialog box in which personal preferences can be set
175. sensitive Spectroscopy for the Life Sciences Bee ere re ope RAS a Chirascan Series User Manual 5 8 6 The Window menu Shotophysics Chirascan Examples Lysozyme Spectra Ly Window Help Mew Window Shift W Close Ctrl Shift W Cascade Tile Arrange Icons 1 Circular Dichroism 2 Absorbance Windows Figure 5 39 the Window menu On the Window menu Figure 5 39 e New Window up a list from which can be selected the desired property The new window will be displayed at the same time as the original window in a tile format e Close will close the currently active window e Cascade will display in a cascade format all property windows e g CD absorbance HT that have been opened and are not currently minimized e Tile will display in a tiled format all property windows e g CD absorbance HT that have been opened and are not currently minimized e Arrange icons will tidy up any minimized property displays that have been strewn around the display window e 1 2 etc refer to the open windows the displayed window is identified by the tick icon e Windows opens a dialog box that enables you to activate or close displays selected from a list 5 8 7 The Help menu The Help menu Figure 5 40 gives access to the ProData help system sics Chirascan Examples Lysozyme Spectra Ly Help Topics About Pro Data Viewer Figure 5 40 the Help menu Document 4207Q108C03 01 Page 109 of 133 Applie
176. sidual noise is to all intents and purposes identical to the single background residual 0 5 0 5 0 5 0 5 Circular Dichroism mdeg Circular Dichroism mdeg 180 190 200 210 220 230 240 250 260 480 190 200 210 220 230 240 250 260 VVavelength nm VVavelength nm Figure 5 43 single background corrected residuals left and their average right Document 4207Q108C03 01 Page 111 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eae tren ee a Chirascan Series User Manual The improvement in S N inherent in averaging is lost and worse any artifact in the background would be reinforced in just the same way as the random noise is and there is thus a danger that any such feature could be misinterpreted as real In order to avoid the impractical and tedious business of measuring a separate background for every spectrum smoothing the background prior to subtracting it from a series of spectra will eliminate the problem A background should not have any sharp features in it and can therefore be smoothed considerably without introducing any artifacts if it does have features it is likely that the cell was not cleaned properly or some other artifact has crept into the measurement and it should be discarded and measured again Repeating the above procedure with a single smoothed background subtracted from the spectra prior to calculating the residuals takes us back to the correct result
177. signal processing firmware to be upgraded via the computer 6 2 Chirascan Windows software Communication between the computer and instrument is carried out over a high speed USB link A purpose designed PCI interface card and driver handles the bi directional communications and data capture 6 2 1 Signal processing Data smoothing during spectral acquisition is provided by user variable digital signal averaging This is applied to the incoming digitised signals at each wavelength of measurement Each resulting averaged data point which may be the result of many thousand individual samples will represent accurately the raw input signal value since all samples are collected at the same wavelength A dedicated high speed averaging algorithm in the driver software carries out the necessary accumulations and computes the averaged result for all monitored signals The operator is able to select the overall sample size This is most usefully presented as the time spent at each spectral wavelength time per point This time will have a direct bearing on the overall scan time and the signal to noise ratio of the result but will never cause a systematic distortion of the kind that can be caused by the use of analog filtering 6 2 2 Application software architecture Figure 6 1 shows the architecture of the spectrometer application software The object oriented design has several benefits particularly from the points of view of maintenance reliability and
178. small magnitude of AA to be measured typically 10 to 10 AU and the high degree of photon shot noise in regions of the spectrum where light throughput is limited This means that signal integration averaging over time is essential and scan times are typically of the order of several minutes particularly when extending into the far UV The selection of a time per point is almost always going to be dictated by the adequacy of the noise reduction in this region of the spectrum and not by the less demanding requirements of regions of the spectrum where there is plenty of light almost everywhere else The result is that in some circumstances in order to have adequate noise improvement in the far UV much time will be spent averaging data elsewhere in the spectrum where it is not necessary The purpose of the adaptive sampling option is to increase scanning efficiency by dynamically altering the data averaging sample size according to the light throughput as the scan proceeds This unique feature of the Chirascan series spectrometers is made possible by variable real time digital signal averaging 6 4 2 The AutoPM function During a normal CD scan the monochromator moves to successive points in the wavelength range of the scan At each position the CD signal is optimised by tuning the gain on the detector by changing the applied high voltage In the spectrometer software the AutoPM function performs this feedback control It operates by increasi
179. so 0_5mm PLOOO4 dsa gt jam ite Lyso 0_5mm PLOOOS dsa tea Lyso 0_5mm PLOOOS dsa tea Lyso 0_5mm PLOOO dsa e Lyso 0_5mm PLOOOS dsa ea Lyso 0_5mm PLOOOS dsa EA R 3 Methylcyclopentanone dsa Alcohol dehydrogenase dsa vfa Bovine serum albumin dsa lt Camphor sulphonic acid dsa Cytochrome C dsa tea Lysozyme dsa tea Trisfethylenediamine cobalt dsa ca Vitamin B12 dsa Manuals F ONW Widgets Ha APLEventMessage dll h 4 mW G Q 1 E z g O L L g a L a U 0 Time Stamp Thu Nov 24 15 42 33 File ID Is CFR Compliant false i Original unaltered data 210 220 230 v VVavelength nm Ready On line CAP NUM Figure 5 1 Pro Data Viewer showing the launchpad left and graphical display right Document 4207Q108C03 01 Page 85 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE ee ERE OS Chirascan Series User Manual 5 1 1 Launching Pro Data Viewer Pro Data Viewer can be launched as a stand alone program using the desktop icon Q and will open at the current working directory Pro Data Viewer will be offline when launched in this way i e there will be no link with the spectrometer control panel SCP Pro Data Viewer can also be launched from the Pro Data Chirascan spectrometer control panel SCP by clicking on the Pro Data Viewer launch icon in the SCP toolbar eo when a client server link between the SCP and Pro Data Viewe
180. srnrrrenrrrrrrenrnrrrnene 113 FIOLO SE A A A A EEA AEE A EA EEA A 113 9 10 2 Using CONN with your own TESUINS ssc scevsicvnweussaswarecdenvenceagiawncecavecsenddhveneed ouvacetsiwnemereuseattinewerbasensaianent 115 5 10 3 Dealing with Uncharacterised Proteins cccccccccsssccccesseccceescccseneccceececssecesseuscesaueesseaesessaueeesaaeeeesees 118 IN OY esos ects ontesa ous nents anne and arsananiapeevansesaananiecasdantauaananeaiaedansensanannunnedamiusgracseoteionswagnantsietes 119 Document 4207Q108C03 01 Page 14 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences sanantseneatenerrsst ener Chirascan Series User Manual 6 1 Key technical features cccccecccccsseeceeceeeeeeceeseceeseseeeseaeeeeeeeeeeeesaaeeesseaeeeessaueeeseageeessaeeeeeseneeeeseeseeessageeseas 119 BA TVS SUD YSTEMS crac sacri nnr EEEE RERE E EE TRET 119 SHAO ELE AN Et aU T A E E E A A A ee ion E artic N A A E EN E E T teat 120 6 1 3 The LAAPD detector Chirascan plus only cccccccccssssssecceceecseessececcceseeeeeececeeeesueeseeeeeesseueaseeceeeseeeegees 121 6 1 4 Control and acquisition electronics ss sssseenessssersssererosserersssrrresssreressererossereeessreresssreresserrressereeesseeeees 123 6 2 Chirascan WIndows SOlWANG wirievsdncimaisancimavamincaaiacsuan EE EEE EEE 124 EL SI A D rOCESSINE erna i E EE OR E OE ARRS 124 6 2 2 Application software architecture sesrsrruscrosrsecrosrsoncirantassinsabiesrnani noiai iusan n
181. st is recorded at the foot of the list Document 4207Q108C03 01 Page 65 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE ee eee Chirascan Series User Manual Skip scans are in general used on samples that you know well and that you might wish to measure again in the future Use the Save As button to save the list as a conf configuration file that can be retrieved using the Load bution at a future date This obviates the need to generate a new list every time you run the sample With the list generated click on OK to return to the SCP When you click on Acquire in the Sequencer panel the monochromator will drive to the wavelengths specified in the list in the order in which they appear in the list and the instrument will measure the chosen property CD voltage fluorescence etc 4 5 The Trigger and Timebase Panels Trigger Timebase intemal Time s 0 05 Logarithmic Paints 400 z C Split Timebase Pressure hold Samples 5 a 45 us Adv Figure 4 11 the Trigger and Timebase panels The Trigger and Timebase panels Figure 4 11 are visible when operating in Kinetics or Spectra Kinetics modes The Trigger drop down menu will only allow the selection of Internal unless the CS SF stopped flow accessory Is fitted The acquisition Time and number of data Points are set using the drop down menus in the Timebase section The Samples box displays the number of samples which will be averaged in order to
182. store containing wavelength as the dimension and CD detector HV absorbance if measured temperature the detector signal voltage DC component and the number of samples taken count as properties lf a temperature ramp is performed in which a series of spectra are measured while the temperature is changing in a single experiment a two dimension Datastore would result with wavelength and temperature as dimensions and containing CD HV etc as properties A titration experiment in which a spectrum is measured at each of a series of concentrations would also generate a two dimension Datastore in this case containing properties as functions of wavelength and titrant concentration Higher dimension Datastores can also be generated If a stepped temperature ramp is carried out using the CS PCM 4 cell autochanger then cell position becomes a third dimension and the Datastore contains properties as functions of temperature wavelength and cell position Substituting time for temperature would result in a Datastore containing properties as functions of wavelength time and cell position All data from a particular experiment are written to a single Datastore Subsequently the Datastore can be mined for subsets of the data Note that the Datastore file format is designed for Pro Data multi dimension data acquisition and is not suitable for direct access outside the Pro Data environment A range of file export options is provided within Pro Dat
183. t may not be known Fortunately that does not prevent you from determining the secondary structure components It is sufficient to replace the molecular weight of the protein with the average molecular weight of the amino acid residues generally accepted to be about 113 and to replace the actual number of amino acids with the number 1 Figure 5 54 CD Signal Type Please indicate type of data units Be carefull Wrong assignment will result in incorrect data renormalization CD Signal Type Essential Parameter C Delta Epsilon Molec mass Dal LE Concentr mg ml 0 200 No amino acids f Mill Degrees Pathlength cm 0 050 mo C Molar Ellipticity Figure 5 54 the CD Signal Type dialog box for an uncharacterised protein Click OK and Deconvolute to generate an identical result to that using the correct molecular weight and the correct number of amino acid residues Document 4207Q108C03 01 Page 118 of 133 Applied hotophysics Ultrasensitive Spectroscopy for the Life Sciences 6 TECHNICAL OVERVIEW 6 1 Key technical features 6 1 1 The subsystems The key technical features of the Chirascan spectrophotometer are e Dual polarising prism optics with digital drive and calibration e High throughput F 7 optical coupling e Rapid and economical nitrogen purge design e High performance digital signal processing of raw data e Expandable modular electronics e High speed real time control e Client server Windows soft
184. tact with any part of the spectrometer Do not clean the spectrometer with organic solvents Use only a soft cloth and water or a mild detergent solution It is good practice to load the sample into the sample cell before the cell is installed in the cell holder Document 4207Q108C03 01 Page 7 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences PEE a eee eee Chirascan Series User Manual CHRASCAN SERIES INSTALLATION AND OPERATIONAL REQUIREMENTS Environmental requirements The spectrometer is best installed in a safe position in a clean air conditioned laboratory environment Operating conditions Temperature 20 C to 25 C controlled to within 1 5 C Humidity 20 to 80 non condensing Dust levels minimum Storage conditions Temperature 20 C to 50 C Humidity 5 to 80 non condensing Bench space The spectrometer with CS PCS Single Cell Temperature Accessory or CS PCM 4 cell Autochanger CS TT Titration Accessory and CS SEM Scanning Emission Monochromator Accessory requires a bench space of Length x depth x height 2 2 x 0 8 x 0 5 metres A sturdy stable vibration free work surface is recommended Electrical requirements The spectrometer is supplied with a filtered mains distribution board which requires an earthed grounded mains electricity supply of between 85 and 285 V AC 50 60 Hz 1000 W maximum Applied Photophysics recommends the use of an uninterruptable power supply UPS rated to 1500 VA
185. tained To acquire a background spectrum click the Background button on the Signal panel Figure 4 4 The background spectrum must have a point corresponding to each point in the measured spectrum it may have additional points It is not necessary to take a background spectrum if only the CD measurement Is required On the Chirascan in CD mode the absorbance is derived from the detector HV and although it gives a good indication of the correct value it may differ slightly from an accurately determined value obtained when the measurement is made in direct absorbance mode on Chirascan or on a dedicated UV visible absorbance spectrometer On the Chirascan plus in CD mode the absorbance is derived from the detector gain which is know precisely and the absorbance is therefore identical to that measured in direct absorbance mode on the Chirascan plus or on a dedicated UV visible spectrometer Unless the spectrometer is equipped with a fluorescence accessory the Fluorescence check box will be inactive See the fluorescence accessories manual for further information on fluorescence measurement The Background subpanel is used to measure a CD background Clicking on Background will initiate a CD measurement using the experimental parameters defined elsewhere in the interface Pro Data viewer will be launched automatically and the display will show the CD background trace The measurement can be stopped at any time by clicking Stop which is the label t
186. teceuposeudauitscueneansiennantaeccaiaenandeunt tees 37 2 8 2 Selecting Spectral Bandwidth SBW cccccsssssscccccccceesseecccceceeeesseceeeeeseuuessececeeeeeeeaeeeeeeeeeseueeseeeeeeeeeaegees 38 2 8 3 Selecting pathlength and CONCENtrAatION ccsccccsssscccesecccceescccceneceeeeueceseuscesseuecessaeseessuecessauseeseaeeessaases 38 284 Selecine the iiime De PON ee wd or en ee ee eee eee 39 ZB Meas ring prole SpecT a 2 aaccsenaplesainaanavacaeaphsacnanstssemeanbiaasianstuseanmaunannauscuscauenuesemeapiianammenpaboanivacanamaaamantnas 39 2 O Example SO CCU a esaa E RE E EEE R EE a E E R E aS 40 29 1 AlCONO GENYGlOSENGSE punso ainera san E E T E TEE E TRE 40 2 9 2 BOVINE serum AIDUMIN ccccceecccceseccccescccaeeeceseuececseuecessueeeseueceesaueeeseesceseuecesseuecessusesssuecesseusessaaeseesaaees 41 Fe EOC NO E a A E E E EE A E EE E ter E E 42 EAN ON O a E E A E E sacutoarearacsescundeaetes 43 29 S VVC BUD e A A E E eaa asian eeesoetnianotetee 44 2 9 6 Tris ethylenediamine cobalt chloride ssseessssseesrrnsesrrrrsesrrrreesrrrressrrrressrrrrssrtressrtresrrtressrreressrrrreseeerene 45 297 Lampr S WPNNI AC ersi a E E et me heer freee en Terre 46 2 9 8 R 3 methylecyclopentanone sess sseancconddecasstnaseannndseusdansaonddonsdeceiedeetediandanncduaedaudsanddecetodtaeinamundscasdaaswanccondeeaed 47 2 10 TrouUDIESNOOUNO faeces saonenesecec ssn sena aedeemensadena ea AAEE eR eE RAEE EREE a AEE RRDA 48 SHARDWAR RE
187. tion that can irritate the eyes and may impair eyesight Never look directly at the light source Do not open the lamp housing while the lamp is operating or immediately after it is powered off Do not allow the skin to be exposed to UV radiation The interaction between ultraviolet light and oxygen leads to the formation of ozone a very reactive gas that is damaging to health and may cause deterioration of the optical components of the instrument If an ozone producing lamp is used it essential that the spectrometer is thoroughly purged with nitrogen before the lamp is powered on WARNING The spectrometer may be equipped with a light source 150 watt xenon or mercury xenon arc leya the electronic circuitry used in the spectrometer is very sensitive and must be correctly grounded to avoid electrical interference It is the user s responsibility to ensure that a proper earth connection can be made Document 4207Q108C03 01 Page 6 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE i ERT SP Chirascan Series User Manual eyd 9 The lamps used in the spectrometer operate at high pressure and can explode or break without warning causing damage to the interior of the lap housing This is more likely to happen with lamps with longer usage and lamps with more than 1000 hours usage should be replaced Corrosive chemical and organic solvents can cause damage to the spectrometer Do not allow corrosive fluids to come into con
188. tory Preferences Help Windows Lysozyme O52 dex C Program Files 86 Lysozyme Spectra E a ete ole Figure 5 25 the launchpad toolbar fet Defines the currently selected directory folder as the working directory i e the directory in which any T new data files will be stored The current working directory is always shown in the box immediately above the toolbar icons The directory name will sometimes need to be contracted to fit in the box but the first and last part of the name are always shown a Returns you to the current working directory Moves the working directory up one level to enable you to access files in directories higher up the tree Creates a new folder in the currently selected directory Opens a browser window from which you can navigate quickly and easily to other folders M L Refreshes the directory tree display This should be done after saving a file following averaging or smoothing for example as the live directory is not automatically updated Click this icon and then on item of interest to call up the context sensitive help for that item y Document 4207Q108C03 01 Page 101 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences Ree E R Chirascan Series User Manual 5 6 2 Graphical display toolbar icons The graphical display toolbar is shown in Figure 5 26 nll Mtr Imi l if EBS amp Ww A l le Gi amp E ae CE A GR a CS e CD
189. ware which supports remote monitoring of experiments in progress e LAAPD based detector system Chirascan plus Monochromator OPTICAL TRAIN Lamp power supply Expansion acquisition Expansion Control modules control module eg modules mono fluorescence detection module temperature sensors digital 1 0 ELECTRONICS RACK USB link Spectrometer control panel handling Chirascan Series User Manual cD detector cD acquisition and PEM control module Power Supplies x2 Figure 6 1 Schematic of the relationship between the Chirascan subsystems Document 4207Q108C03 01 Page 119 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences eee Deen ee Chirascan Series User Manual The schematic diagram of spectrometer in Figure 6 1 illustrates the relationship between the three subsystems of the instrument the optical train the control and acquisition electronics and the spectrometer Windows software The optical train is described more fully in Section 6 1 2 of this Chapter the control and acquisition electronics in Section 6 1 4 and the Windows software in Chapters 4 and 5 6 1 2 Optical train A schematic of the Chirascan series spectrometer optical train is shown in Figure 6 2 Monochromator Lamp housing Sample handling unit Detector Detector M1 focussing mirror M2 M5 folding mirrors S1 S3 slits P1 P2 prisms L1 L2 lenses PEM photoelastic modulator
190. ws one of which lists any traces that are visible in the graphics window the other listing those that have been removed from the display during the session and are no longer shown in the display The buttons on the right execute actions on traces that have been highlighted in the Visible Traces window Traces can be swapped between the Removed Traces and Visible Traces window using the arrow buttons between the two panes Single arrow will move a selection double arrow will move all listed traces In the example below there are six traces 3 repeat backgrounds and 3 repeat lysozyme spectra We will average the backgrounds average the spectra subtract the average background from the average spectrum smooth the resulting background corrected lysozyme spectrum and remove the original data The end result will show the lysozyme spectrum and its residual calculated minus observed spectrum With the data loaded into the display as previously described and the graphical display active open the dialog box to display the data files list Figure 5 10 E C Program Files x86 Applied Photophysics Chirascan Examples Lysozyme Spectrallyso O_Smm PLOODM dsa Cieculae Dichroism gy ag File e Trace Math Scale Wind Heir x S0aN 2 oe 15 10 Circular Dichroism mdeg 180 190 200 210 220 230 240 250 260 VVavelength nm Figure 5 10 raw data with dialog box called up Document 4207Q108C03 01 Page 92 of 133 Applied
191. y 1 mg ml can therefore be determined accurately from its absorbance characteristics A solution containing exactly 1 mg ml will have an absorbance of 0 1486 AU ina 10 mm pathlength cuveite Using proportionality one can then calculate the CD value for a CSA solution once its absorbance at 285 nm as been accurately determined 1S CSA is available from the Sigma Aldrich Company product code C 1395 1S 10 camphorsulphonic acid Sigma C 1395 Molecular weight 232 3 Target concentration 1 mg ml Distilled water Table 4 1 conditions for CD calibration Document 4207Q108C03 01 Page 82 of 133 Applied Ultrasensitive Spectroscopy for the Life Sciences FE eRe Ee Chirascan Series User Manual The conditions for the calibration are summarized in Table 4 1 Firstly make up a solution of CSA at about 1 mg ml in distilled water Determine its concentration accurately by measuring its absorbance maximum at 285 nm ina 10 mm pathlength cell It is important to use the Absorbance program Section 4 2 3 to calculate the absorbance of the CSA see also Chapter 6 of this User Manual The concentration is calculated from Concentration of CSA mg ml Observed absorbance 0 1486 and the expected CD value for this concentration in a 10 mm pathlength cell at 290 5 nm thus CD value at 290 5 nm 335 x Observed absorbance 0 1486 Set the wavelength to 290 5 nm the bandwidth to 1 nm and the time per point to 1 second If you have administrator p
192. ysics Chirascan Examples Lysozyme Spectra Lyso 0_5mm PL0005 dsa Circular D 0 File View Directory Preferences Help E File View Trace Math Scale Window Help X Windows Lyso 0_ Smm PLOO05 dsa hSOervr OHO O8OH8 00 9 0 0 8 a n eS J E a CK ae applied Photophysics L Chirascan H ADDUSERS E 15 Config Examples Ge Lysozyme Spectra a Lyso O_5Smm PLOOO4 dsa tea Lyso 05mm PLOOOS dsa A Lyso O_5mm PLOOO dsa Lyso O_5mm PLOOO dsa Lyso O_5mm PLOOOS dsa fa Lyso 0_5mm PLOOOS dsa A Rj 3 Methylcyclopentanone dsa 3 Alcohol dehydrogenase dsa 3 Bovine serum albumin dsa 3 Camphor sulphonic acid dsa Cytochrome C dsa 3 Lysozyme dsa Trisfethylenediamine cobalt dsa Vitamin B12 dsa Manuals H ONW i Widgets APLEventMessage dll Re 4 P m File Lyso 0_5mm PL0008 dsa ProDataStore Attributes Time Stamp Thu Nov 24 15 42 33 File ID Is CFR Compliant false A A A Original unaltered data 180 190 200 210 220 230 240 250 260 Remarks Circular Dichroism mdeg m Se VVavelength nm 4 m Ready On line Ready CAP NUM Figure 2 11 Pro Data Viewer showing the launchpad left and graphical display right 2 5 1 Viewing spectra With the Datastores Explorer open navigate in the usual way until you find the file of interest and double click on it Pro Data viewer displays the data contained i

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